Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.     

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.     

Effect of exogenous CDP-choline on choline metabolism in isolated adult rat ventricular myocytes under normoxic and hypoxic conditions

The purpose of this study was to examine the effect of exogenous CDP-choline on choline metabolism and phosphatidylcholine biosynthesis in adult rat ventricular myocytes. Choline uptake and metabolism were examined, using [methyl³ H] choline. CDP-choline in the medium produced a concentration dependent reduction in the amount of radio-label in phosphocholine and phospholipid but it did not alter choline uptake into the myocytes. CDP-choline also did not antagonize the effect of hypoxia on phosphatidylcholine synthesis; rather it accentuated the hypoxia-induced reductions in cellular phosphocholine and phosphatidylcholine biosynthesis. These results indicate that the exogenous administration of CDP-choline alters choline metabolism in the heart by reducing the formation of phosphocholine and phosphatidylcholine without altering choline uptake and suggest an effect of a CDP-choline metabolite on choline metabolism which is not effective in opposing the effect of hypoxia on phosphatidylcholine biosynthesis.     

Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells.

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.     

胆碱摄取及磷脂酰胆碱合成的调节研究

木文研究了多种氨基酸、乙醇胺和甲基乙醇胺对细胞摄取胆碱和合成磷脂酰胆碱（ＰＣ）的影响,发现多种氨基酸非竞争性地抑制细胞摄取胆碱。含胆碱代谢物的分析显示胆碱转变成ＣＤＰ－胆碱,随之形成ＰＣ均不受氨基酸影响。乙醇胺竞争性地抑制胆碱摄取,且存在剂量依赖关系。乙醇胺能明显抑制胆碱激酶活性,但细胞内胆碱和磷酸胆碱的代谢池并不改变,提示乙醇胺不影响胆碱转变成磷酸胆碱。根据ＣＤＰ－胆碱和ＰＣ的比放射性分布,乙醇胺也不影响ＰＣ的生物合成。甲基乙醇胺抑制胆碱摄入的程度强于乙醇胺,并抑制胆碱激酶和ＣＴＰ：磷酸胆碱胞苷转移酶活性,含胆碱代谢物以ＣＤＰ－胆碱下降最显著;提示甲基乙醇胺不仅抑制胆碱摄入而且还干扰了ＣＤＰ－胆碱通路。     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.     

Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters.

It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.     

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.     

Uptake and metabolism of choline in the organotypic culture of newborn mouse cerebellum

The uptake and metabolism of [methyl-14C]choline in the organotypic culture of newborn mouse cerebellum was examined. Explants of 8 day in vitro (8 DIV) were incubated for 48 h under standard conditions with 21.0 microM [14C]choline at 35 degrees C. During the first hour of incubation, most of the [14C]choline incorporated was transferred to phosphocholine. The amount of [14C]phosphocholine increased gradually at the initial rate of 0.95 +/- 0.17 nmol/mg protein/h and saturated after 7 h (4.31 +/- 1.30 nmol/mg protein). The synthesis of [14C]phospholipids was observed after a distinct time lag. About 96% of the radioactivity in the lipids was incorporated into phosphatidylcholine. The amount of phosphatidylcholine increased linearly up to 48 h of incubation: 11.9 +/- 2.10 nmol/mg protein at 24 h and 21.9 +/- 2.43 nmol/mg protein at 48 h. From double-label studies it was found that phosphocholine was a precursor of phosphatidylcholine. The content of [14C]choline within explants remained nearly constant through the incubation period. Acetylcholine synthesis in mouse cerebellum culture was relatively low, and the content remained constant through the incubation period (0.006 +/- 0.003 nmol/mg protein). Activities of acetylcholine synthesis of cerebral and cerebellar homogenates were compared. Phosphatidylcholine synthesized in mouse cerebellum culture separated into two spots on thin layer chromatograph using silica gel G plates. Gas chromatographs suggested that the separation depends on the difference in fatty acid composition.     

Ethanolamine inhibits choline uptake in the isolated hamster heart

The effect of exogenous ethanolamine on phosphatidylcholine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [Me-3H]choline in the presence of 0.05-0.5 mM ethanolamine. Incorporation of label into phosphatidylcholine was decreased 26-63% at 0.1-0.5 mM ethanolamine. Similar decreases in the labelling of the metabolites of the CDP-choline pathway were observed at these ethanolamine concentrations. The observed decrease in phosphatidylcholine labelling at 0.1-0.5 mM ethanolamine was attributed to an inhibition of labelled choline uptake by ethanolamine. The inhibitory role of ethanolamine to choline uptake was examined by comparison to hemicholinium-3. Both compounds inhibited choline uptake in a competitive manner. Intracellular choline, phosphocholine and CDP-choline concentrations were not altered under all experimental conditions. It can be concluded that exogenous ethanolamine has no immediate effect on the rate of phosphatidylcholine biosynthesis in the isolated hamster heart. The reduced labelling of phosphatidylcholine in the presence of ethanolamine is a direct result of the reduction of labelled choline taken up by the heart.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

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 phosphatidylethanolamine, 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. J. Cell. Biochem, 64:196–208. © 1997 Wiley-Liss, Inc.     

Choline Synthesis in Spinach in Relation to Salt Stress

Choline metabolism was examined in spinach (Spinacia oleracea L.) plants growing under nonsaline and saline conditions. In spinach, choline is required for phosphatidylcholine synthesis and as a precursor for the compatible osmolyte glycine betaine (betaine). When control (nonsalinized) leaf discs were incubated for up to 2 h with [1,2-14C]ethanolamine, label appeared in the N-methylated derivatives of phosphoethanolamine including phosphomono-, phosphodi-, and phosphotri- (i.e. phosphocholine) methyl-ethanolamine, as well as in choline and betaine, whereas no radioactivity could be detected in the mono- and dimethylated derivatives of the free base ethanolamine. Leaf discs from salinized plants showed the same pattern of labeling, although the proportion of label that accumulated in betaine was almost 3-fold higher in the salinized leaf discs. Enzymes involved in choline metabolism were assayed in crude leaf extracts of plants. The activites of ethanolamine kinase and of the three S-adenosylmethionine:phospho-base N-methyltransferase enzymes responsible for N-methylating phosphoethanolamine to phosphocholine were all higher in extracts of plants salinized step-wise to 100, 200, or 300 mM NaCI compared with controls. In contrast, choline kinase, phosphocholine phosphatase, and cytidine 5[prime]-triphosphate: phosphocholine cytidylyltransferase activities showed little variation with salt stress. Thus, the increased diversion of choline to betaine in salt-stressed spinach appears to be mediated by the increased activity of several key enzymes involved in choline biosynthesis.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.     

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.     

Intraperitoneal administration of CDP-choline and its cholinergic and pyrimidinergic metabolites induce hyperglycemia in rats: involvement of the sympathoadrenal system

CDP-choline is an endogenous metabolite in phosphatidylcholine biosynthesis. Exogenous administration of CDP-choline has been shown to affect brain metabolism and to exhibit neuroprotective actions. On the other hand, little is known regarding its peripheral actions. Intraperitoneal administration of CDP-choline (200-600 micromol/kg) induced a dose- and time-dependent hyperglycemia in rats. Hyperglycemic response to CDP-choline was associated with several-fold elevations in serum concentrations of CDP-choline and its metabolites. Intraperitoneal administration of phosphocholine, choline, cytidine, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, uridine, uridine monophosphate, uridine diphosphate and uridine triphosphate also produced significant hyperglycemia. Pretreatment with atropine methyl nitrate failed to alter the hyperglycemic responses to CDP-choline and its metabolites whereas hexamethonium, the ganglionic nicotinic receptor antagonist which blocks nicotinic cholinergic neurotransmission at the autonomic ganglionic level, blocked completely the hyperglycemia induced by CDP-choline, phosphocholine and choline, and attenuated the hyperglycemic response to cytidine monophosphate and cytidine. Increased blood glucose following CDP-choline, phosphocholine and choline was accompanied by elevated plasma catecholamine concentrations. Hyperglycemia elicited by CDP-choline and its metabolites was entirely blocked either by pretreatment with a nonselective -adrenoceptor antagonist phentolamine or by the 2-adrenoceptor antagonist, yohimbine. Hyperglycemic responses to CDP-choline, choline, cytidine monophosphate and cytidine were not affected by chemical sympathectomy, but were prevented by bilateral adrenalectomy. Phosphocholine-induced hyperglycemia was attenuated by bilateral adrenalectomy or by chemical sympathectomy. These data show that CDP-choline and its metabolites induce hyperglycemia which is mediated by activation of ganglionic nicotinic receptors and stimulation of catecholamine release that subsequently activates 2-adrenoceptors.     

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.     

CPT-cAMP and okadaic acid enhance phosphatidylcholine catabolism in choline-deficient rat hepatocytes.

The effect of CPT-cAMP and okadaic acid on phosphatidylcholine catabolism in suspension cultures of choline-deficient rat hepatocytes was investigated. Choline-deficient hepatocytes were pulse-labeled for 30 min with [methyl-3H]choline and subsequently chased for up to 60 min with choline in the absence or presence of 0.5 mM CPT-cAMP or 0.5 microM okadaic acid. Radioactivity in phosphatidylcholine and lysophosphatidylcholine were unchanged during the chase. However, the radioactivity incorporated into glycerophosphocholine was significantly increased (P less than 0.05) 59 and 77% after 60 min of chase in hepatocytes incubated with either okadaic acid or CPT-cAMP, respectively. Incubation of choline-deficient hepatocytes with both okadaic acid and CPT-cAMP produced an additive effect on radioactivity incorporated ino glycerophosphocholine. Crude mitochondrial, microsomal, and cytosolic phospholipaselysophospholipase activities, assayed in the presence of exogenously labeled phosphatidylcholine, were unchanged in both CPT-cAMP and okadaic acid treated hepatocytes compared with control. Phospholipase-lysophospholipase activity, assayed with endogenously labeled phosphatidylcholine, was increased 28 and 47% (P less than 0.05) in the crude mitochondrial fraction of hepatocytes treated with either okadaic acid or CPT-cAMP, respectively, compared with the control. Incubation of choline-deficient hepatocytes, labeled with L-[methyl-3H]methionine, with CPT-cAMP or okadaic acid caused a 31 and 20% increase (P less than 0.05) in the radioactivity incorporated into glycerophosphocholine, respectively, compared with the control. We postulate that phosphatidylcholine catabolism in choline-deficient hepatocytes may be regulated by a phosphorylation-dephosphorylation mechanism mediated through cAMP-dependent protein kinase and phosphoprotein phosphatase activities.