Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action

Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane.     

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.     

Regulation of ethanolamine and choline phosphatide biosynthesis in isolated rat intestinal villus cells

The effects of ethanolamine, choline, and different fatty acids on phospholipid synthesis via the CDP-ester pathways were studied in isolated rat intestinal villus cells. The incorporation of [14C]glucose into phosphatidylethanolamine was stimulated severalfold by the addition of ethanolamine and long-chained unsaturated fatty acids, while the addition of lauric acid inhibited the incorporation of radioactivity into phosphatidylethanolamine. At concentrations of ethanolamine higher than 0.2 mM, phosphoethanolamine accumulated, but the concentrations of CDP-ethanolamine and the incorporation of radioactivity into phospatidylethanolamine did not increase further. The incorporation of [14C]glucose into phosphatidylcholine responded in a way similar to that of phosphatidylethanolamine, except that a 10-fold higher concentration of choline was required for maximal stimulation. CCC inhibited the incorporation of choline into phosphatidylcholine. In contrast with hepatocytes, villus cells did not form phosphatidylcholine via phospholipid N-methylation. The data indicate that, in intestinal villus cells, the cytidylyltransferase reactions are rate limiting in the synthesis of phosphatidylethanolamine and probably also of phosphatidylcholine. The availability of diacylglycerol and its fatty acid composition may also significantly affect the rate of phospholipid synthesis.     

The phospholipid and fatty acid composition of skeletal muscle cells during culture in the presence of vitamin D-3 metabolites

The phospholipid and fatty acid composition of primary cultures (24 h) of chick embryo skeletal muscle myoblasts treated for 4-24 h with physiological concentrations of 1,25-dihydroxyvitamin D-3 and 25-hydroxyvitamin D-3 were analyzed. 25-Hydroxyvitamin D-3 did not alter the relative amounts of individual muscle cell phospholipids whereas 1,25-dihydroxyvitamin D-3 significantly increased phosphatidylcholine content, mainly at the expense of a decrease in phosphatidylethanolamine concentration. The increase in phosphatidylcholine occurred at a faster rate during the first 8 h than in the subsequent 8-24 h treatment period. A similar time course in 1,25-dihydroxyvitamin D3-dependent changes in myoblast calcium uptake has been observe. In addition, this metabolite markedly increased (100%) the arachidonate content of myoblast phosphatidylcholine near the fusion stage of the cells (24 h of treatment). The levels of docosahexaenoate, a minor polyunsaturated fatty acid, in phosphatidylcholine and phosphatidylethanolamine were also substantially elevated by 1,25-dihydroxyvitamin D-3. No significant changes in fatty acid composition in response to 25-hydroxyvitamin D-3 were observed. Modifications in phospholipids and polyunsaturated fatty acids may play a role in the effects of 1,25-dihydroxyvitamin D-3 on muscle cell calcium transport and differentiation.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

Induction of specific proteins in cultured skeletal muscle cells by 1,25-dihydroxyvitamin D-3

The presence in myoblasts of an intracellular receptor specific for 1,25-dihydroxyvitamin D-3 [1,25(OH)2D3) and 1,25(OH)2D3-dependent changes in myoblast Ca2+ transport and phospholipid metabolism which are suppressed by RNA and protein synthesis inhibitors have been shown. In agreement with these observations, incubation of chick embryo myoblasts, precultured for 24 h in a medium containing low levels of vitamin D-3 metabolites, with 1,25(OH)2D3 at conditions which induce maximum cell responses (10(-10) M, 24 h) markedly stimulated the incorporation of [3H]leucine into total cell proteins and this effect was abolished when sterol treatment was performed in the presence of cycloheximide or puromycin. To investigate whether 1,25(OH)2D3 selectively stimulates the de novo synthesis of muscle cell proteins, mixtures of myoblast proteins from control and sterol-treated cultures labelled with [14C]leucine and [3H]leucine, respectively, were separated by SDS-polyacrylamide gel electrophoresis and isoelectric focussing. Examination of 3H/14C ratios in gel fractions revealed that 1,25-(OH)2D3 stimulates the production of proteins of molecular masses (isoelectric points) of 9 kDa (4.1 and 8.5), 17 kDa (7.5), 30 kDa (7.2), 40 kDa (5.5), 55 kDa (4.5) and 100 kDa (8.6). Cell fractionation studies showed the following subcellular distribution: 9 kDa (85% cytosol, 15% microsomes); 17 and 100 kDa (100%, 1200 X g pellet); 30 kDa (65% cytosol, 35% mitochondria); 40 kDa (100% microsomes); 55 kDa (65% microsomes, 35% mitochondria). Marker enzyme data indicated that this distribution is not due to cross-contamination between fractions. Affinity chromatography of double-labelled myoblast proteins on an immobilized lectin showed that the 55 kDa protein contains carbohydrate. Labelling of myoblast proteins with 45CaCl2 after their separation on SDS-polyacrylamide gels showed in addition that the 1,25(OH)2D3-dependent proteins of 9, 17, 40 and 100 kDa are major Ca2+-binding components of the cells. Synthesis of these proteins may mediate the effects of the sterol on myoblast calcium metabolism.     

Palmitoyl-L-carnitine, a metabolic intermediate of the fatty acid incorporation pathway in erythrocyte membrane phospholipids

In this paper we report that palmitoyl-L-carnitine can be a metabolic intermediate of the fatty acid incorporation pathway into erythrocyte membrane phosphatidylcholine, and phosphatidylethanolamine. Phospholipid acylation was evaluated by measuring the incorporation of radioactive [1-14C]-palmitoyl-L-carnitine in membrane erythrocyte ghost phospholipids in the presence or absence of CoA. CoA highly stimulated the incorporation of [1-14C]-palmitic acid into both the phospholipids examined, although the incorporation was also evident in the absence of added CoA. Incorporation of [1-14C]-palmitic acid into phosphatidylcholine was greater than into phosphatidylethanolamine. 2-Bromo-palmitoyl-CoA, an irreversible inhibitor of the erythrocyte carnitine palmitoyltransferase, inhibited the acylation process.     

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.     

The apparent transfer of fatty acid from phosphatidylcholine to phosphatidylethanolamine in human erythrocytes

In previous studies an apparent transfer of (14)C-labeled fatty acid from phosphatidylcholine to phosphatidylethanolamine was observed in prelabeled human erythrocytes reincubated in fresh serum. These data could have been explained by direct fatty acid transfer from phosphatidylcholine to phosphatidylethanolamine or by an apparent transfer simulated by either demethylation of labeled phosphatidylcholine to phosphatidylethanolamine or base-exchange of phosphatidylcholine with ethanolamine. To explore these possibilities, RBC containing phosphatidylcholine doubly labeled with palmitic acid-9,10-(3)H and with choline-1,2-(14)C were prepared. Upon reincubation in fresh serum, incorporation of (3)H (fatty acid) into phosphatidylethanolamine was observed without incorporation of (14)C (choline). In similar experiments in which RBC labeled with (3)H-labeled fatty acid alone were used, (14)C-ethanolamine added to the incubation was not incorporated into the isolated phosphatidylethanolamine which again showed incorporation of the fatty acid-(3)H. The data indicate that direct transfer of fatty acid from phosphatidylcholine to phosphatidylethanolamine can occur in human erythrocytes incubated in fresh serum.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

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.     

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

A deazaadenosine-insensitive methylation of phosphatidylethanolamine is involved in lipoprotein secretion

We have examined the effect of inhibitors of methylation of phosphatidylethanolamine on lipoprotein secretion from cultured rat hepatocytes. The incorporation of [1-3H]ethanolamine into phosphatidylcholine of hepatocytes and secreted lipoproteins was inhibited by greater than 90% by the methylation inhibitors 3-deazaadenosine and Neplanocin. In addition, these compounds strongly inhibited the incorporation of [3-3H]serine into the choline moiety of phosphatidylcholine of the hepatocytes, but had no effect on incorporation of [3-3H]serine into secreted phosphatidylcholine. The results suggest that a pool of phosphatidylcholine targeted for lipoprotein secretion originates from phosphatidylethanolamine made from serine and this methylation reaction has the unique property of being insensitive to 3-deazaadenosine.     

Glycerolipid metabolism in lung from ventilated and unventilated rabbits

The incorporation of [14C]-glycerol 3-phosphate and [3H]-palmitate into phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and triacylglycerols by lung microsomes from ventilated and unventilated rabbits was measured. Unventilated lung microsomes showed an impairment of the "de novo" synthesis of phosphatidic acid and, therefore, a general decrease of glycerolipids synthesized from glycerol 3-phosphate. The incorporation of [3H]-palmitate into phosphatidic acid was considerably lower than the incorporation of [14C]-glycerol 3-phosphate by lung microsomes from both ventilated and unventilated rabbits, and the 3H/14C molar ratio did not change during incubation time. These observations suggest the preferential utilization of endogenous fatty acids by acyltransferases involved in the formation of phosphatidic acid. The activities of the enzymes implicated in the synthesis of phosphatidylcholine from lysophosphatidylcholine remained unchanged in lung from both ventilated and unventilated rabbits.     

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.     

Role of a specific choline pool in sphingomyelin synthesis in rat heart.

Sphingomyelin synthesis was studied in slices of rat heart by using [Me-14C]choline, [1,2-14C]ethanolamine, S-adenosyl-L-[14C]methionine and [32P]Pi as as precursors. In the presence of both [Me-14C]choline and [32P]Pi the ratio of the specific radioactivities of 14C and 32P in phosphatidylcholine was greater than in sphingomyelin at all the times studied. This suggested that synthesis of phosphatidylcholine and sphingomyelin de novo did not involve the utilization of a common pool of cytidine diphosphate choline. In addition, studies with [1,2-14C]ethanolamine and S-adenosyl-L-[14C]methionine indicated that a quantitatively significant pool of choline, derived from these precursors, was selectively utilized for sphingomyelin formation. This pool was not represented by phosphatidylcholine formed by methylation of phosphatidylethanolamine or by other pathways.