Metabolism, mitochondrial uptake and toxicity of 2', 3'-dideoxycytidine.
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2',3'-Dideoxycytidine (ddCyd) is a prescription anti-retroviral drug that causes mitochondrial toxicity and peripheral neuropathy. ddCyd is actively phosphorylated by cytosolic deoxycytidine kinase and nucleoside (di)phosphate kinase to the 5'-triphosphate derivative. However, 2',3'-dideoxycytidine 5'-diphosphocholine (ddCDP-choline) was also found in human cells incubated with ddCyd. In this paper we show that ddCDP-choline is produced from dideoxyCTP (ddCTP) and phosphocholine by phosphocholine cytidylyltransferase. dCTP and CTP appear to activate this synthesis in a concentration-dependent manner. Although ddCTP and ddCDP-choline can both enter the mitochondria, ddCDP-choline uptake is more efficient than ddCTP uptake. These data suggest that ddCDP- choline is the ddCyd metabolite that is probably responsible for mitochondrial toxicity. The uptake of ddCTP and ddCDP-choline by mitochondria is inhibited by 3.0 mM l-carnitine in the cell-free system investigated; when added to U937 cells grown in the presence of 0.25 microM ddCyd, 3.0 mM l-carnitine partially abrogated the mitochondrial toxicity of ddCyd. (+info)
One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity.
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In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane. (+info)
Effects of intratracheal instillation of TNF-alpha on surfactant metabolism.
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Tumor necrosis factor-alpha (TNF-alpha) has been shown to play an integral role in the pathogenesis of the acute respiratory distress syndrome. This disorder is characterized by a deficiency of alveolar surfactant, a surface-active material that is composed of key hydrophobic proteins and the major lipid disaturated phosphatidylcholine (DSPC). We investigated how TNF-alpha might alter DSPC content in rat lungs by instilling the cytokine (2.5 microg) intratracheally for 10 min and then assaying parameters of DSPC synthesis and degradation in alveolar type II epithelial cells, which produce surfactant. Cells isolated from rats given TNF-alpha had 26% lower levels of phosphatidylcholine compared with control. TNF-alpha treatment also decreased the ability of these cells to incorporate [(3)H]choline into DSPC by 45% compared with control isolates. There were no significant differences in the levels of choline substrate or choline transport between the groups. However, TNF-alpha produced a 64% decrease in the activity of cytidylyltransferase, the rate-regulatory enzyme required for DSPC synthesis. TNF-alpha administration in vivo also tended to stimulate phospholipase A(2) activity, but it did not alter other parameters for DSPC degradation such as activities for phosphatidylcholine-specific phospholipase C or phospholipase D. These observations indicate that TNF-alpha decreases the levels of surfactant lipid by decreasing the activity of a key enzyme involved in surfactant lipid synthesis. The results do not exclude stimulatory effects of the cytokine on phosphatidylcholine breakdown. (+info)
Hepatocyte growth factor is elevated in chronic lung injury and inhibits surfactant metabolism.
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Adult respiratory distress syndrome may incorporate in its pathogenesis the hyperplastic proliferation of alveolar epithelial type II cells and derangement in synthesis of pulmonary surfactant. Previous studies have demonstrated that hepatocyte growth factor (HGF) in the presence of serum is a potential mitogen for adult type II cells (R. J. Panos, J. S. Rubin, S. A. Aaronson, and R. J. Mason. J. Clin. Invest. 92: 969-977, 1993) and that it is produced by fetal mesenchymal lung cells (J. S. Rubin, A. M.-L. Chan, D. P. Botarro, W. H. Burgess, W. G. Taylor, A. C. Cech, D. W. Hirschfield, J. Wong, T. Miki, P. W. Finch, and S. A. Aaronson. Proc. Natl. Acad. Sci. USA 88: 415-419, 1991). In these studies, we expand on this possible involvement of HGF in chronic lung injury by showing the following. First, normal adult lung fibroblasts transcribe only small amounts of HGF mRNA, but the steady-state levels of this message rise substantially in lung fibroblasts obtained from animals exposed to oxidative stress. Second, inflammatory cytokines produced early in the injury stimulate the transcription of HGF in isolated fibroblasts, providing a plausible mechanism for the increased amounts of HGF seen in vivo. Third, HGF is capable of significantly inhibiting the synthesis and secretion of the phosphatidylcholines of pulmonary surfactant. Fourth, HGF inhibits the rate-limiting enzyme in de novo phosphatidylcholine synthesis, CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15). Our data indicate that fibroblast-derived HGF could be partially responsible for the changes in surfactant dysfunction seen in adult respiratory distress syndrome, including the decreases seen in surfactant phosphatidylcholines. (+info)
Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells.
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PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis. (+info)
Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase.
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We investigated the effects of tumor necrosis factor alpha (TNFalpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFalpha significantly inhibited [(3)H]choline incorporation into PtdCho after 24 h of exposure. TNFalpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNFalpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCTalpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of MLE cells. Northern blotting revealed no effects of the cytokine on steady-state levels of CCTalpha mRNA, and CCTbeta mRNA was not detected. Incorporation of [(35)S]methionine into immunoprecipitable CCTalpha protein in pulse and pulse-chase studies revealed that TNFalpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTalpha. Addition of N-acetyl-Leu-Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNFalpha. TNFalpha-induced degradation of CCTalpha protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFalpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFalpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways. (+info)
Functional significance of Sp1, Sp2, and Sp3 transcription factors in regulation of the murine CTP:phosphocholine cytidylyltransferase alpha promoter.
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The transcription factor Sp1 has been implicated in regulation of the expression of the murine CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene, Ctpct (M. Bakovic, K. Waite, W. Tang, I. Tabas, and D. E. Vance. 1999. Biochim. Biophys. Acta. 1438: 147;-165). We have utilized transient transfections, mutation analysis, electromobility gel-shifts, and immunoblot analysis to test the hypothesis that expression of the CTalpha gene is controlled in part by the binding of three trans-acting nuclear factors, Sp1, Sp2, and Sp3. Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains. In Sp1-mediated transcription, Sp3 acts as an activator in a dose-dependent manner and vice versa. Sp2 represses Sp1- and Sp3-driven transcription in Drosophila SL2 cells, but stimulates transcription in C3H10T1/2 mammalian cells. Our results suggest that the predominant action of Sp proteins is a direct function of local organization of three cis-acting elements in the regions A (-31/-9), B (-88/-50), and C (-148/-128). The ability of distal C (-148/-128) and proximal A (-31/-9) regions to activate or repress transcription depends upon the cellular background. The multiple binding elements at position B (-88/-50) confer a positive regulation independent of the cell context. However, the effectiveness of Sp proteins at this site is strongly governed by neighboring sites A and C. The results suggest that the level of expression of the CTalpha gene will depend on the cell type, the availability of Sp proteins, and the structure and organization of three cis-acting elements. (+info)
Regulation of phosphatidylcholine metabolism in Chinese hamster ovary cells by the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein pathway.
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Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4-6-fold increase in [(3)H]choline incorporation into PtdCho and 10-15-fold increase in intracellular [(3)H]glycerophosphocholine. [(3)H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase alpha (CCTalpha). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [(3)H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [(3)H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTalpha protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTalpha in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTalpha to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTalpha by fatty acids or a fatty acid-derived signal. (+info)