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.     

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.     

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.     

Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were found to provoke increases in [3H]2-deoxyglucose uptake, diacylglycerol (DAG) generation and membrane-bound protein kinase C activity in BC3H-1 myocytes. These effects were similar to those provoked by insulin. The increases in DAG did not appear to be derived from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol, but may have been derived from synthesis of phosphatidic acid de novo, and hydrolysis of phosphatidylcholine, as revealed by studies with [3H]glycerol and [3H]choline respectively. Accordingly, both EGF and IGF-I increased acute [3H]glycerol labelling of DAG (and other lipids) and [3H]choline labelling of phosphocholine. These labelling responses were similar in time course, suggesting that they are closely coupled. Our findings suggest that EGF and IGF-I, like insulin, increase DAG-protein kinase C signalling, apparently by activating co-ordinated lipid-synthesis and -hydrolysis responses, which are distinctly different from the PIP2-hydrolysis response.     

Production of diacylglycerol by exogenous phospholipase C stimulates CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in human neuroblastoma cells.

The involvement of endogenous diacylglycerol production in the stimulation of phosphatidylcholine synthesis by exogenous phospholipase C was examined using a neuroblastoma (LA-N-2) cell line. Phospholipase C treatment (0.1 unit/ml) of intact cells stimulated CTP:phosphocholine cytidylyltransferase activity significantly more effectively than did maximally effective concentrations of the synthetic diacylglycerol sn-1,2-dioctanoylglycerol (1 mM). When added to cells together with phospholipase C, oleic acid, but not dioctanoylglycerol, further increased cytidylyltransferase activity with respect to phospholipase C treatment alone, indicating that the enzyme was not maximally activated by the lipase. This suggests that the lack of additivity of diacylglycerol and phospholipase C reflects a common mechanism of action. The time course of activation of cytidylyltransferase by phospholipase C paralleled that of [3H]diacylglycerol production in cells prelabeled for 24 h with [3H]oleic acid. Diacylglycerol mass was similarly increased. Significant elevations of [3H]oleic acid and total fatty acids occurred later than did the increases in cytidylyltransferase activity and diacylglycerol levels. No significant reduction in total or [3H]phosphatidylcholine was elicited by this concentration of phospholipase C, but higher concentrations (0.5 unit/ml) significantly reduced phosphatidylcholine content. The stimulation of cytidylyltransferase activity by phospholipase C or dioctanoylglycerol was also associated with enhanced incorporation of [methyl-14C]choline into phosphatidylcholine. Dioctanoylglycerol was more effective than phospholipase C at stimulating the formation of [14C]phosphatidylcholine, and the effects of the two treatments were additive. However, further analysis revealed that dioctanoylglycerol served as a precursor for [14C]dioctanoylphosphatidylcholine as well as an activator of cytidylyltransferase; and when corrections were made for this effect, the apparent additivity disappeared. The results indicate that the generation of diacylglycerol by exogenous phospholipase C (and possibly the subsequent production of fatty acids via diacylglycerol metabolism) activates cytidylyltransferase activity in neuronal cells under conditions in which membrane phosphatidylcholine content is not measurably reduced.     

Hormonal regulation of phosphatidylcholine synthesis by reversible modulation of cytidylyltransferase.

The effect of both lipolytic and antilipolytic hormones on the turnover of phosphatidylcholine in freshly isolated rat adipocytes was investigated. Treatment of adipocytes with agonists such as glucagon or isoprenaline that stimulate lipolysis through a cyclic AMP-dependent mechanism caused an increase in the incorporation of [Me-3H]choline into phosphatidylcholine. Pulse-chase studies indicated that the stimulation was due to an increase in the conversion of choline into phosphatidylcholine, which was both time- and dose-dependent. The stimulatory effect of isoprenaline was inhibited in a dose-dependent manner by oxytocin or insulin. Oxytocin inhibited the incorporation of [Me-3H]choline into phosphatidylcholine in both the presence and the absence of isoprenaline, whereas in the absence of isoprenaline insulin increased the incorporation of [Me-3H]choline into phosphatidylcholine. The effects of isoprenaline, oxytocin and insulin on the incorporation of [3H]choline into phosphatidylcholine were paralleled by changes in the activity of CTP:phosphocholine cytidylyltransferase.     

Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.     

Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown

Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with [3H]glycerol or [3H]choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in [3H]ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 [3H])phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.     

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.     

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

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

Phosphatidylcholine Metabolism in Nuclei of Phorbol Ester-Activated LA-N-1 Neuroblastoma Cells

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuro-blastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [³H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [³H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPS. The stimulation of [³H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.     

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.     

Bradykinin stimulates phosphodiesteratic cleavage of phosphatidylcholine in cultured endothelial cells

The ability of bradykinin to stimulate phosphodiesteratic cleavage of phosphatidylcholine (PC) was investigated in bovine pulmonary artery endothelial cells prelabeled with [3H]choline and [3H]myristic acid. Both labels were preferentially (approximately 80%) incorporated into PC. Bradykinin stimulated a rapid and parallel increase in approximately equivalent amounts of water soluble ([3H]choline plus [3H]phosphocholine) and lipid ([3H]phosphatidic acid plus [3H]diacylglycerol) phosphodiesteratic cleavage products of PC. Formation of the phosphodiesteratic cleavage products occurred prior to the maximum rate of release of prostacyclin into the medium, and ED50 values for both responses were similar (less than 1 nM) and consistent with effects mediated by a high affinity bradykinin receptor. These findings suggest that phosphodiesteratic cleavage of PC may be an important event in the process of receptor-dependent endothelial cell activation.     

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.     

Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies

Biosynthesis of sphingomyelin from ceramides was investigated in lung subcellular fractions by incubating a lyophilized mixture of albumin and subcellular fraction (0.1-0.2 mg of protein) coated with [acyl-14C]-ceramide and phosphatidyl[methyl-3H]choline in Tris-buffer. The lamellar-body-rich fraction exhibited the highest specific activity for sphingomyelin biosynthesis measured by 14C incorporation into sphingomyelins or by [3H]phosphocholine transfer from phosphatidylcholines. Plasma membranes formed the next most active fraction, followed by the 'smooth' and, then, the 'rough' endoplasmic reticulum. Sphingomyelin biosynthesis by lamellar bodies was optimum at pH 7.4 and was inhibited by sphingomyelins formed. Slight inhibitory effects were also observed with Mn2+, Ca2+ and lysophosphatidylcholine. Phosphocholine transfer from CDPcholine was not observed under the reaction conditions employed. Ceramide conversion and phosphocholine transfer increased with ceramide concentration to reach a maximum at about 0.06 mM. The highest conversion rate was observed when 18:1 ceramide was used as an acceptor. When 1-palmitoyl-2-oleoylphosphatidylcholine was the phosphocholine donor, the overall biosynthesis of sphingomyelin was much higher than when using dipalmitoylphosphatidylcholine. These results suggest the possible involvement of the studied reaction in the control of the degree of saturation of the surfactant phosphatidylcholine.     

Antigen-induced biphasic diacylglycerol formation in RBL-2H3 cells: the late sustained phase due to phosphatidylcholine hydrolysis is dependent on protein kinase C

Exposure to antigen (Ag) caused a biphasic 1,2-diacylglycerol (DG) production in [3H]myristic acid-labeled RBL-2H3 cells; the early, small transient phase and the second large sustained phase. The accumulation of phosphatidic acid (PA) or phosphatidylethanol (PEt) in the presence of ethanol was paralleled by the second-phase DG generation. Ag-induced formation of phosphocholine and choline in [3H]choline-labeled cells suggested the hydrolysis of phosphatidylcholine (PC) by phospholipases C and D. Treatment with phorbol myristate (PMA) or A23187 caused increases in [3H]DG and water-soluble [3H]choline metabolites. In protein kinase C (PKC) down-regulated cells, PEt formation was markedly reduced. In these cells DG production induced by Ag and A23187 was largely suppressed, thus indicating that PKC would play an important regulatory role for PC hydrolysis. However, because the A23187 treatment showed significant accumulation of water-soluble choline metabolites in PKC down-regulated cells, an increase in intracellular Ca2+ is another factor regulating PC hydrolysis. Taken together, these results may indicate that PC hydrolysis in response to Ag is dependent on PKC and Ca2+.     

Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.     

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.