Increase In Choline Incorporation Research Articles

Identification of sex-specific differences in surfactant synthesis in rat lung.

Delayed lung maturation and lower levels of surfactant phosphatidylcholine have been previously identified in male fetuses compared with female fetuses in several species. We investigated the mechanisms for sex differences in surfactant content by examining parameters of phosphatidylcholine turnover and biosynthesis; the latter was evaluated by measuring metabolic steps within the biosynthetic pathway. Compared with male lung cells, freshly isolated lung cells from female fetuses contained higher levels of disaturated phosphatidylcholine, a marker of surfactant lipid. Female mixed monolayer cultures exhibited a 71% increase in choline incorporation into disaturated phosphatidylcholine compared with male cultures. Male cultures exhibited significantly greater release of [3H]-arachidonic acid into the medium compared with females, suggesting sex differences in phospholipase activity. However, pulse-chase studies showed no sex differences in degradation of disaturated phosphatidylcholine, which was confirmed by assays of phospholipase A2, phosphatidylcholine-specific phospholipase C, and phospholipase D. Female mixed lung cells, however, had greater rates of cellular choline transport and activity of cytidylyltransferase, the rate-regulatory enzyme for phosphatidylcholine synthesis. Separate studies showed that exposure of sex-specific pretype II cell cultures to cortisol-stimulated fibroblast-conditioned medium plus transforming growth factor-beta-neutralizing antibody stimulated cytidylyltransferase activity to a greater extent in male cells compared with female cells. These studies indicate that sex differences in surfactant phospholipid content are not due to differences in phospholipid turnover, but rather differential regulation of specific metabolic steps within the surfactant biosynthetic pathway. The data also support a role for transforming growth factor-beta as a negative regulator of a key surfactant biosynthetic enzyme within male lungs.

Cystic fibrosis and phosphatidylcholine biosynthesis

The cystic fibrosis (CF) gene defect may be associated with a defect in membrane recycling. We have investigated the metabolism of the main constituent of plasma membrane, phosphatidylcholine (PC). In this study of platelets and fibroblasts, we show an increased uptake of choline into PC of CF cells as compared with normal cells. No accumulation of PC was seen. Other patients with respiratory disease (not CF) showed normal rates of incorporation of choline into platelet PC. Platelets from heterozygote individuals showed intermediate turnover rates of choline incorporation into PC. The increase in choline incorporation into PC in CF platelets was not due to modified or increased sensitivity to either cAMP or prostaglandin E 2. The total amount and the proportions of the major phospholipids in platelets of control and CF individuals were identical. These findings indicate an increased turnover rate of this phospholipid in CF cells rather than an increased net synthesis.

Reduction of phosphatidylcholine turnover in a Nb 2 lymphoma cell line after prolactin treatment. A novel mechanism for control of phosphatidylcholine levels in cells.

Phosphatidylcholine metabolism was investigated in Nb 2 rat node lymphoma cells, a cell line which is dependent on prolactin for growth in culture. Treatment of stationary cultures with prolactin stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine (1.7-fold after 4 h) and its aqueous precursors, mainly phosphocholine (1.9-fold after 4 h and 2.7-fold after 10 h). These effects were blocked by cycloheximide. Pulse-chase studies demonstrated that the reaction catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) was rate-limiting for phosphatidylcholine synthesis in Nb 2 cells and that the rate of this reaction was not altered by prolactin treatment. The cell-free activity of choline kinase (EC 2.7.1.32) was found to increase in correspondence with the increase in choline incorporation. This induction of choline kinase was also blocked by cycloheximide. The activities of the other enzymes of phosphatidylcholine synthesis were unchanged. These results suggest that phosphatidylcholine biosynthesis was not altered in Nb 2 cells after prolactin treatment. However, phosphatidylcholine levels increased in prolactin-treated cells (1.4-fold after 16 h). Turnover of labeled phosphatidylcholine was markedly reduced in prolactin-treated cells. Calculated turnover rates for phosphatidylcholine averaged 4.2-fold lower in prolactin-treated cells, whereas the synthetic rates were similar in prolactin-treated and stationary cells. Thus, Nb 2 cells utilize a novel mechanism, reduction of turnover, to regulate the cellular levels of phosphatidylcholine during growth.

Thyroid hormone stimulation of phosphatidylcholine synthesis in cultured fetal rabbit lung.

To investigate the mechanism of thyroid hormone action on pulmonary surfactant synthesis, we characterized the effect of triiodothyronine on phosphatidylcholine synthesis in cultured fetal rabbit lung. Since glucocorticoids stimulate surfactant synthesis and reduce the incidence of Respiratory Distress Syndrome in premature infants, we also examined the interaction of triiodothyronine and dexamethasone. The rate of choline incorporation into phosphatidylcholine was determined in organ cultures of rabbit lung maintained in serum-free Waymouth's medium. In 23-d lung cultured for 72 h, the increase in choline incorporation with triiodothyronine alone, dexamethasone alone, and triiodothyronine plus dexamethasone was 50, 62, and 161%, respectively. Both triiodothyronine and dexamethasone also increased incorporation rates with glucose, glycerol, and acetate as precursors, and stimulation with triiodothyronine plus dexamethasone was at least additive. Dexamethasone, but not triiodothyronine, affected distribution of radioactivity from [3H] acetate among phospholipids. Stimulation was first detected 8-12 h after addition of triiodothyronine, and then increased in a linear fashion. With triiodothyronine plus dexamethasone, stimulation was maximal at 48-72 h, and was supra-additive at all times. Exposure of cultured lung to dexamethasone enhanced the subsequent response to triiodothyronine, but not vice versa. When triiodothyronine was removed from cultures, there was no further stimulation and the triiodothyronine effect was partially reversed within 24 h. Half-maximal stimulation of choline incorporation occurred at a triiodothyronine concentration (0.10 nM) very similar to the dissociation constant for triiodothyronine binding to nuclear receptor (0.11 nM). The relative potencies of thyroid hormone analogs for nuclear binding and stimulation of phosphatidylcholine synthesis were also similar: triiodothyroacetic acid greater than triiodothyronine-proprionic acid greater than L-triiodothyronine approximately D-triiodothyronine much greater than thyroxine much greater than 3,5-diethyl-3'-isopropyl-DL-thyronine approximately 3,5-dimethyl-3'-isopropyl-L-thyronine approximately reverse triiodothyronine. The effect of triiodothyronine was blocked by the presence of either actinomycin D or cycloheximide, inhibitors of ribonucleic acid and protein synthesis, respectively. We conclude that triiodothyronine stimulates phosphatidylcholine synthesis by a process involving nuclear receptors and de novo ribonucleic acid and protein synthesis. These findings support the concept that endogenous triiodothyronine has a physiologic role in lung maturation and suggest that a combined antenatal therapy with thyroid hormone and glucocorticoid may be useful for prevention of Respiratory Distress Syndrome in the premature infant.

Fetal rat lung maturation: initiation and modulation.

The purpose of this study was to determine whether a rise in the level of circulating hormones is responsible for initiating fetal lung maturation. Explants of 13- to 20-day fetal rat lung were cultured in a constant chemically defined medium containing 0 or 1% serum. Growth, morphological maturation, and two biochemical markers of lung development, disaturated phosphatidylcholine (DSPC) synthesis and specific nuclear glucocorticoid binding, showed evidence of continuing development in culture. There is normally a marked increase in DSPC content and in the rate of choline incorporation into DSPC after 20 days gestation. Regardless of the gestational age of the fetal lungs used, there was a similar increase in culture at an equivalent gestational age of 20 days (e.g., 14-day lung after 6 days, 16-day lung after 4 days). Removal of 50 or 75% of the lung mesenchymal tissue at the initiation of the culture period did not prevent the increase in choline incorporation. Since the culture environment was constant and contained virtually no hormones, we conclude that the stimulus for the initiation of fetal lung maturation is most likely located in the lung tissue itself. The role of circulating hormones is probably later modulation of the maturational process.

Alveolar pre-type II cells from the fetal rabbit lung isolation and characterization

A method for preparing a homogeneous population of undifferentiated cells from the fetal rabbit lung is described. This method utilizes enzymatic digestion, differential adhesion to remove fibroblasts and centrifugation on a discontinuous metrizamide gradient. Cells isolated by this procedure replicate in vitro in medium supplemented with carbon-stripped fetal bovine serum. Mitosis can also be stimulated by heat-inactivated medium conditioned by fetal lung fibroblasts. After confluence, exposure of these cells to 0.55 or 55 nM dexamethasone significantly increased the incorporation of [ 14C]choline into phosphatidylcholine. Lower concentrations of the drug also increased incorporation, but not significantly so. Addition of heat-inactivated fibroblast-conditioned medium produced a 25% increase in choline incorporation, but this was not significant. Furthermore, the presence of conditioned medium tended to reduced the response of the cells to dexamethasone. After confluence, lamellar inclusion bodies were present in more than 90% of those cells exposed to dexamethasone. These organelles were not observed in cell monolayers not exposed to the steroid. These cells did contain a few small electron-dense bodies. The latter may be immature multivesicular bodies.

Phospholipid synthesis by activated human B lymphocytes.

Pokeweed mitogen- (PWM) stimulated DNA and Ig synthesis in human B cells is dependent on the presence of T cells and adherent cells, but the influence of these regulatory cells on earlier activation events is unknown. We have studied the T cell and monocyte influence on the incorporation of [methyl-14C]choline chloride into B cell phospholipids (PL) after varying periods of in vitro culture with or without pokeweed mitogen (PWM). By separating B and T cells after choline pulsing, a peak in PWM-induced PL synthesis of B cells at days 1 to 2 was revealed, whereas the T cell response was later (days 2 to 3). In the first 4 hr of culture, the purified B cell plus monocyte fraction incorporated choline four to six times faster than the T cell fraction, but PWM did not increase choline incorporation, whether these fractions were cultured separately or together. When cultures were pulsed with choline between 16 and 20 hr with or without PWM, monocytes incorporated choline six to nine times faster than T cells, and B cells were intermediate. Also at 16 to 20 hr of culture, a significant PWM-induced increase in choline incorporation by B cells was evident and was dependent on the presence of T cells and monocytes. The monocytes showed no increased choline incorporation due to PWM. Thus, the influence of regulatory cells on the PWM response in B cells is evident within the first 24 hr.

Renotrophic factors in urine

Our previous observations of increased renal protein synthesis in rats subjected to the constant intravenous reinfusion of half their urine output has suggested that the circulatory retention of renotrophic factors in urine is capable of stimulating renal growth. In the present studies, using this same model of "half-urine-reinfusion," which is designed to produce a selective halving of renal excretory function, we have demonstrated significant increases in total DNA content and the incorporation of tritiated thymidine in renal DNA. In addition, a bioassay method was developed in which an assay rat, given an intravenous infusion of urine from another rat, exhibited increases in the incorporation of thymidine into renal DNA and the incorporation of radiolabelled choline into renal phospholipid. This renotrophic activity in the urine was only minimally decreased by heating to 100 degrees C for 30 min and was confined to ultrafiltration fractions retained on a membrane with a nominal 10,000-dalton solute rejection. Removal of one kidney from the rats from which the urine was obtained led to only a modest and transient reduction in the excretion of renotrophic activity, suggesting that the urinary renotrophic factors are of circulatory, not renal, origin. Isolated renal cortical fragments incubated with an ultrafiltration retentate of urine displayed a dose-dependent increase in choline incorporation into phospholipid, suggesting a direct action of the factors on kidney tissue. Finally, no evidence of stimulation of either DNA or phospholipid synthesis could be seen in hepatic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a regulatory role of CTP : Choline phosphate cytidylyltransferase in the synthesis of phosphatidylcholine in fetal lung following premature birth

The sequence of reactions which function to incorporate choline into phosphatidylcholine was investigated in lung from fetuses following premature delivery. The rate of [ methyl- 14C]choline incorporation by rat lung slices into phosphatidylcholine increases following premature delivery at both 20 and 21 days gestation. The increase in choline incorporation is primarily due to an increased specific activity of phosphorylcholine resulting from a decreased pool size of phosphorylcholine. The decrease in the concentration of phosphorylcholine following premature delivery is apparently caused by an increased activity of cytidylyltransferase which leads to an increase in the conversion of phosphorylcholine to phosphatidylcholine. The total activity of choline kinase, cytidylyltransferase, cholinephosphotransferase and phosphatidate phosphohydrolase did not change significantly. However, the cytidylyltransferase activity in the microsome fraction increased following premature delivery at 20 and 21 days gestation. The amount of cytidylyltransferase in the H form in the cytosol fraction increased following premature delivery at 21 days gestation but not at 20 days gestation. The results are interpreted to indicate that the active form of cytidylyltransferase in lung cells is the membrane-bound enzyme and this form increases following birth resulting in an increased synthesis of phosphatidylcholine.

Aminophylline stimulates the incorporation of choline into phospholipid in explants of fetal rat lung in organ culture

We have investigated the direct effect of aminophylline, a cyclic AMP phosphodiesterase inhibitor, and of cyclic AMP on choline incorporation into phospholipid in explants of fetal rat lung in organ culture. Explants from 19 day fetal rat lung (term is 22 days) were cultured for 24 or 48 h in F12 medium containing 1.0 mM aminophylline or 0.2 mM dibutyryl cyclic AMP. Explants cultured in F12 medium alone served as controls. Exposure to aminophylline resulted in an 88% increase in choline incorporation into phosphatidylcholine ( P < 0.01) and a 95% increase in choline incorporation into sphingomyelin ( P < 0.001) in the 48-h cultures. Similar changes were noted after 24 h in culture. There was a small increase in the percentage of synthesized phosphatidylcholine which was disaturated. The activities of choline kinase and cholinephosphotransferase in the explants were not significantly altered by exposure to aminophylline. 77 and 53% stimulation of choline incorporation into phosphatidylcholine and sphingomyelin respectively was demonstrated in explants cultured in medium containing cyclic AMP. Aminophylline treatment of explants from 20- and 21-day fetuses resulted in a smaller degree of stimulation of choline incorporation into both phospholipids. These data suggest that aminophylline has a direct stimulatory effect on the incorporation of choline into phospholipid in fetal rat lung and that this effect is probably cyclic AMP mediated.

Glucocorticoids and fetal lung development

Acceleration of fetal lung development by administration of glucocorticoid hormones has been demonstrated in a number of mammalian species. Dexamethasone 10 −5 M significantly depressed protein content per explant in the presence and absence of insulin. Insulin did not alter the protein content but significantly increased radioactive choline incorporation. However, dexamethasone provided no added stimulation above that observed with insulin alone. The incorporation of radioactive choline and specific activities in isolated phosphatidylcholine were almost identical in control and dexamethasone (10 −7 M) treated explants. The data presented demonstrate a modest but significant increase in choline incorporation using a concentration of dexamethasone of 10 −9 M. At higher concentrations of dexamethasone, the incorporation was significantly depressed using an 8 day exposure time. Although choline incorporation increases as a function of gestational age with a burst in rate on the last day of fetal life, dexamethasone suppresses this activity at all developmental ages past 19.5 days. The only conclusion that appears valid at this time is the rat fetal lung system reported here differs in an unknown way when compared to the rabbit monolayer system, the human organ culture, and the in vivo situation.

Inhibition of Echovirus-12 multiplication by N-carbobenzoxy-D-glucosamine.

The glucosamine derivative, N-carbobenzoxy-D-glucosamine (NCBZG) inhibits the multiplication of Echovirus-12 and the synthesis of both virus RNA and protein at a stage in the virus growth cycle after attachment and penetration. However, the compound does not inhibit virus multiplication after the appearance of progeny virus nor after virus RNA has accumulated. Incorporation of radioactive glucosamine and choline into infected and uninfected cultures is inhibited by NCBZG as is the virus-induced increase in choline incorporation. The compound also prevents the appearance of radioactive choline in isolated membranous structures. The compound did not alter significantly the cellular RNA or protein synthesis, plating efficiency of the cells, their growth over a period of several days, nor the virus-directed inhibition of cellular RNA and protein. These findings suggest that the compound inhibits virus multiplication by its effect on the initiation of biosynthesis which appears to require membrane synthesis.