Integration of phospholipid and sterol metabolism in mammalian cells.

The lethal consequences of imbalances in lipid and sterol metabolism in human diseases such as atherosclerosis and lipid storage disorders underscores our need to know how cholesterol, phospholipid and sphingolipid metabolism is integrated. Accumulation and abnormal localization of lipids and sterol affects cellular function not only by perturbing membrane activity but also by increasing production of bioactive lipids derived from cholesterol, phospholipids and sphingolipids. For example in the NPC mouse model, accumulation of intracellular cholesterol and sphingomyelin is accompanied by increased sphingosine [187], a potent regular of protein kinase C and cell proliferation [152]. Oxidized LDL has an important role in the pathology of atherosclerosis by promoting foam cell formation and cytotoxicity [65]. 7-Hydroxycholesterol and 7-ketocholesterol are involved in many aspects of oxidized LDL activity including initiation of apoptosis in a number of cell types [188, 189] and enhancing cholesterol accumulation by inhibiting efflux [190]. Oxysterols formed intracellularly or from oxidized lipoproteins could have an important role in regulating lipid metabolism in the foam cell. Bioactive metabolites of phospholipids, such as diglyceride, phosphatidic acid and lysolipids, could also increase in circumstances of elevated deposition and have profound and varied effects on cell physiology. In addition to elucidating mechanisms for integration of lipid metabolism, we should determine when these responses go awry and assess the influence of bioactive compounds formed under these circumstances on cell viability and growth.

Incorporation, metabolism and positional distribution of trans-unsaturated fatty acids in developing and mature brain. Comparison of elaidate and oleate administered intracerebrally.

Mixtures of [1-14C]elaidic acid and [9,10-3H]oleic acid, as well as [1-14C]elaidic or [1-14C]oleic acid alone, were administered by intracerebral injection to 10-day old and adult rats to examine the relative incorporation and metabolism of trans- and cis-monounsaturated fatty acids in developing and mature brain. Rates and extents of total incorporation of label from trans- and cis-acid into complex lipids were similar. Maximum labeling of the neutral lipid, mainly triacylglycerol and cholesterol, occurred prior to 4 h after injection whereas phospholipid, mainly choline phosphoglyceride, was maximally labeled at 4--8 h after injection. The decrease in labeled phospholipid from 24 to 90 h was greater with elaidate than with oleate. At 8 h labeled elaidic acid was preferentially esterified in the 1-position of all phosphoglycerides whereas labeled oleic acid, in contrast to the positional distribution of endogenous oleate, was randomly distributed. Label from elaidate found in palmitate increased with time to 26% of the total recovered label by 48 h. Thus, although some of the trans-fatty acid is oxidized and its labeled carbon is redistributed, most is incorporated unaltered into complex lipid of brain at rates similar to those for its cis-isomer. The developing central nervous system, therefore, does not metabolically exclude the trans-acid, elaidic acid, from membrane lipids.

Studies of the modulation of essential fatty acid metabolism by fatty acids in cultured neuroblastoma and glioma cells.

In cultured neuroblastoma cells (N1E-115), the metabolism of the essential fatty acid, linoleic acid (18:2 (n-6)), to arachidonic acid (20:4(n-6)) can be altered by other fatty acids in a manner supporting a concerted action of the modulating fatty acid on the desaturation and chain elongation enzymes. In further examination of mechanisms involved, cultured glioma (C-6) or neuroblastoma-glioma hybrids (NG-108-15) cells showed similar patterns of activation by some fatty acids (e.g., 20:3(n-6) and 20:4(n-6)), and inhibition (e.g., 18:3(n-3) or 22:6(n-3)) or no effect (e.g., 18:1(n-9), 20:3(n-3)) by others. In contrast, only inhibition by 20:4(n-6) was seen in cultured HeLa cells, suggesting that the intracellular interactions may not be universal in all cell lines. For fatty acids that activate 20:4(n-6) formation, the lag observed when substrate and activator were administered simultaneously was eliminated by preincubation with activator. Maximal activation occurred within 4 h for neuroblastoma and 2 h for glioma; in each cell line activation declined steadily for 10 h after removal of the activator. Inhibition of protein synthesis did not alter activation. As 98% of the fatty acid incorporated was esterified to triacylglycerol or phospholipid and only the triacylglycerol mass expanded, several manipulations to potentially alter the flow of acyl chains between these lipid pools were evaluated using dual-label and pulse-chase experiments. Results suggested that competition between 18:2(n-6) utilization for esterification to phospholipid and the desaturation-chain elongation sequence as well as a more direct and specific interaction of certain fatty acids with the enzymes may influence 20:4(n-6) formation. A model to explain these observations is discussed.

Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine.

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.

Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane.

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.

Membrane-bound CTP:phosphocholine cytidylyltransferase regulates the rate of phosphatidylcholine synthesis in HeLa cells treated with unsaturated fatty acids.

The influence of fatty acids on phosphatidylcholine biosynthesis in HeLa cell cultures was investigated. Oleate and other unsaturated fatty acids stimulated the incorporation of phospho[Me-3H]choline into phosphatidylcholine from 5 to 20-fold, while saturated fatty acids were without effect. Stimulation of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) by 1 mM oleate was evident within 15 min and could be reversed within 40 min after removal of the oleate-supplemented cell medium. Cytidylyltransferase activity was 11-fold higher in homogenates from cells exposed to oleate. Treatment of the HeLa cells with oleate produced almost complete translocation of the cytidylyltransferase from the cytosol to the microsomal fraction. Additional support for conversion of the cytidylyltransferase to a membrane-bound form in oleate-treated cells was obtained in studies with digitonin. Exposure of control cells to digitonin for 2 min caused the release of 60% of the total cytidylyltransferase into the medium, while oleate-treated cells leaked only 5% of the enzyme in the presence of digitonin. Finally, oleate (50 microM) was shown to promote complete aggregation of cytosolic cytidylyltransferase with microsomes from HeLa cells and 22-fold stimulation of the enzyme activity. It appears that the rate of phosphatidylcholine biosynthesis is governed by the amount of cytidylyltransferase bound to endoplasmic reticulum in HeLa cells.

Rapid isolation of neuroblastoma plasma membranes on Percoll gradients. Characterization and lipid composition.

A purified plasma membrane fraction was isolated from cultured neuroblastoma (N1E-115) cells on a discontinuous gradient of 5, 25 and 35% Percoll within 1 h of cell disruption by nitrogen cavitation. Yield of plasma membrane, banding in the 25% Percoll (d = 1.051), was high as judged by the recoveries of the marker enzymes, 5'-nucleotidase (58.0 +/- 5.4%, n = 5), alkaline phosphatase (46.0 +/- 3.0%, n = 4) and Mg2+-stimulated neutral sphingomyelinase (48.0 +/- 4.2%, n = 3); enrichment of specific activities of these enzymes relative to total cell homogenate (lysate) were 10.9 +/- 1.0-, 9.1 +/- 1.0- and 9.6 +/- 0.4-fold, respectively. Levels of marker enzymes for other organelles were less than 3% of total activity, except for microsomes (less than 9%). The plasma membrane fraction was further characterized by 2-, 5- and 6-fold higher content (nmol/mg protein) of total phospholipids, free cholesterol and sphingomyelin, respectively, compared to lysate. Ratios of free cholesterol to phospholipids and of sphingomyelin to phosphatidylcholine in the plasma membrane fraction were about 2-fold greater than that of lysate. The cholesterol ester content of plasma membrane (36 +/- 8 nmol/mg protein) was 2-3-fold higher than that of lysate. Sphingomyelin of the plasma membrane fraction had a higher concentration of long-chain fatty acids (more than 18 carbon atoms) relative to lysate or microsomes. Significant differences also were observed in the fatty acyl composition of diphosphatidylglycerol, cholesterol esters and triacylglycerol of plasma membrane. Thus, we have devised a rapid and reliable method for isolation of highly purified plasma membranes of cultured neuroblastoma cells that is suitable for comparison of metabolic relationships between the plasma membrane and other cellular organelles.

Phospholipid transfer protein-mediated incorporation and subcellular distribution of exogenous phosphatidylcholine and sphingomyelin in cultured neuroblastoma cells.

Murine neuroblastoma cells (strain N1E-115) were incubated with 2-[fatty acyl-14 C]acylphosphatidylcholine/sphingomyelin or phosphatidylcholine/[choline-3H]sphingomyelin liposomes (1:1, mol/mol; 1.2 mumol total lipid/mg cell protein) in the presence of partially purified rat liver phospholipid transfer protein (2.5 mg/ml), cytochalasin B (50 microM) and 2-deoxyglucose (50 mM) for 10 min. Washed cells were chased for periods of up to 45 min at 37 degrees C with medium containing transfer protein and unlabeled liposomes. Total transfer protein-dependent incorporation of [14C]phosphatidylcholine ([14C] PC) and [3H]sphingomyelin was 136.7 +/- 26.5(n = 5) and 23.7 +/- 5.4(n = 6) nmol/mg protein per 10 min incubation, respectively, (mean +/- S.D.). Incorporation of [14C]PC into the mitochondrial membrane fraction was 128-fold greater (nmol/mg protein) than incorporation of [3H]sphingomyelin. In contrast, incorporation of [3H]sphingomyelin into a fraction enriched in plasma membrane and into microsomes was 1.4- and 2.6-fold greater, respectively, than incorporation of [14C]PC. During the chase periods, the specific activities of total cellular phospholipids decreased as intact [14C]PC and [3H]sphingomyelin accumulated in the culture medium. In the case of cells labeled with [14C]PC, the effect was due primarily to a decrease in the amount of labeled phospholipid in the mitochondrial fraction; in the case of cells labeled with [3H]sphingomyelin, the decrease in activity was greatest in microsomal and plasma membrane phospholipids. The rate and extent of non-endocytotic incorporation of exogenous phosphatidylcholine into the cell membrane of cultured neuroblastoma cells, and its subsequent subcellular disposition, is different from that of exogenous sphingomyelin. Whereas PC is evidently incorporated into and turned over most rapidly in fraction enriched in mitochondrial membranes, sphingomyelin appears to be preferentially incorporated into microsomal and plasma membrane.

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes.

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.

Activation of phospholipase D by PKC and GTPgammaS in human neuroblastoma cells overexpressing MARCKS.

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.

Effect of fumonisin B1 on phosphatidylethanolamine biosynthesis in Chinese hamster ovary cells.

Fumonisin B1 has been shown to inhibit dihydroceramide synthesis and elevate cellular sphinganine levels in several cultured cell lines. In Chinese hamster ovary (CHO)-K1 cells, 20 microM fumonisin B1 inhibited sphingomyelin synthesis by 75% after 5 h, but stimulated [3H]serine incorporation into PtdEtn by 5- to 7-fold. Fumonisin caused a 10-20% increase in [3H]serine labelling of PtdSer. While fumonisin (20 microM) caused sustained inhibition of sphingomyelin synthesis, PtdEtn labelling peaked at 7-fold above controls at 12 h and declined to 4-fold by 24 h. Fumonisin treatment for 12 h increased the in vitro activity of PtdSer synthase by 62% and inhibited PtdSer decarboxylase by 35%, suggesting that increased PtdEtn labelling by [3H]serine is not by this pathway. An ethanolamine 'trap' experiment was performed to assess the contribution of phosphoethanolamine from sphinganine degradation for PtdEtn labelling. Stimulation of [3H]serine incorporation into PtdEtn by fumonisin could be reduced by 60% with the inclusion of 50 microM unlabelled ethanolamine in the culture medium. The ethanolamine-mediated reduction in [3H]serine incorporation into PtdEtn was accompanied by 4-fold increase in cellular [3H]phosphoethanolamine. In control cells labelled with [3H]serine, 50 microM ethanolamine did not cause [3H]phosphoethanolamine to accumulate. Consistent with elevated phosphoethanolamine production in fumonisin-treated cells, [3H]ethanolamine incorporation into PtdEtn was inhibited by 75% after 12 h. The degradation of endogenous long-chain bases to phosphoethanolamine and entry into the CDP-ethanolamine pathway appears to be a major pathway for PtdEtn synthesis in fumonisin-treated CHO-K1 cells.

Clofibrate and other peroxisomal proliferating agents relatively specifically inhibit synthesis of ethanolamine phosphoglycerides in cultured human fibroblasts.

Effects of several classes of peroxisomal proliferators on peroxisomal functions, hepatomegaly, hepatocarcinogenesis and lipid metabolism have been extensively investigated in rodents. Less is known about influences of these agents, some used as hypolipidemic drugs, on various metabolic parameters in humans. We examined effects of clofibrate, di(2-ethyl-hexyl)phthalate (DEHP) and pirinixic acid (WY-14,643) on phospholipid metabolism in human fibroblasts in culture. Clofibrate inhibited incorporation of [1-14C]hexadecanol and [1-14C]linolenic acid into ethanolamine phosphoglycerides in a time- and concentration-dependent manner; labeling of plasmalogens and non-plasmalogen ethanolamine phosphoglycerides was reduced by 40-80% compared to a generalized 10-30% inhibition of labeling of other phospholipids, including phosphatidylcholine. In pulse and pulse-chase experiments, selective inhibition of incorporation of [1,2-14C]ethanolamine, compared to [methyl-3H]choline, confirmed relative specificity of inhibition of ethanolamine phosphoglycerides. Similar concentration dependence and specificity for inhibition of phospholipid turnover was observed for DEHP and WY-14,643, in both control and mutant (Zellweger and adrenoleukodystrophy) fibroblasts, in the absence of major effects on peroxisomal markers. These observations that peroxisomal proliferators specifically inhibit ethanolamine phosphoglyceride turnover in human fibroblasts should be considered when assessing the efficacy and safety of such agents as hypolipidemic drugs or when evaluating mechanisms of proliferator action at the cellular level.

Thapsigargin selectively stimulates synthesis of phosphatidylglycerol in N1E-115 neuroblastoma cells and phosphatidylinositol in C6 glioma cells.

Phospholipid metabolism was studied in N1E-115 neuroblastoma and C6 glioma cells exposed to thapsigargin, a selective inhibitor of endoplasmic reticulum Ca(2+)-ATPase that raises the cytosolic free Ca2+ concentration [Ca2+]i. Thapsigargin caused only a transient increase of [Ca2+]i (< 1 min) in N1E-115 cells similar in magnitude and duration to agonist-induced calcium release mediated by inositol trisphosphate. Sustained elevation of [Ca2+]i due to influx of extracellular calcium, as occurs in most other cell lines including C6 cells, did not occur in N1E-115 cells. Increased uptake of inorganic phosphate (Pi) associated calcium influx was observed in C6 but not in N1E-115 cells. Thapsigargin affected phospholipid synthesis in both cell lines, most likely by inhibiting phosphatidic acid phosphohydrolase as indicated by diversion of [3H]oleic acid incorporation from triacylglycerol to phospholipid synthesis and stimulation of [32P]Pi incorporation into anionic phospholipids at the expense of phosphatidylcholine synthesis. The response to increased phosphatidate/phosphatidyl-CMP availability was cell specific. Thapsigargin (> 100 nM) selectively stimulated phosphatidylglycerol synthesis 20-30-fold in N1E-115 neuroblastoma cells while phosphatidylinositol synthesis was increased < 2-fold. In contrast, phosphatidylglycerol was not affected in C6 glioma cells and phosphatidylinositol synthesis was stimulated 8-fold by thapsigargin (> 1 microM). Agonist-stimulated calcium release did not increase phosphatidylglycerol synthesis in N1E-115 cells. Thapsigargin-stimulated phosphatidylglycerol synthesis and agonist-stimulated phosphatidylinositol synthesis could occur at the same time. Similar results were obtained with TMB-8, an inhibitor of intracellular Ca2+ release that decreases diacylglycerol utilization by blocking choline uptake and phosphatidylcholine synthesis without affecting resting [Ca2+]i. Thus [Ca2+]i does not directly mediate the effects of thapsigargin, TMB-8 or agonist stimulation on anionic phospholipid metabolism. These additional effects may limit the use of thapsigargin to assess Ca(2+)-dependence of phospholipid metabolism associated with Ca(2+)-mediated signal transduction.

Phosphatidylcholine metabolism in cultured cells: catabolism via glycerophosphocholine.

The catabolism of phosphatidylcholine (PtdCho) has been studied in cultured murine neuroblastoma (N1E-115), C6 glioma, rat brain primary glia, and human fibroblast cells. Cells were pulse labelled for 96 h with [methyl-3H]choline followed by a chase for up to 24 h in medium containing 4 mM choline. Measurement of the radioactivity and mass of choline-containing compounds in these cells indicated that the major degradative pathway is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----choline. At all times during the chase, PtdCho, sphingomyelin and lysoPtdCho comprised 72-92% of the cell-associated radioactivity; the remaining 10-30% was water-soluble and was chiefly GroPCho (30-80%) in all cell lines. In fibroblasts, however, phosphocholine (PCho) was also a major labelled water-soluble component (33-54%). The specific activity of GroPCho closely parallelled that of PtdCho in fibroblasts, but decreased faster than PtdCho in C6 and N1E-115 cells. We postulate that this may be due to distinct pools of PtdCho in the cell with differing rates of turnover. The changes in specific activity of PCho suggest that the major portion is formed by synthesis rather than as a degradative product. However, the inability to reduce the specific activity of this fraction to that of the intracellular choline suggests that a portion may be derived from either PtdCho or GroPCho.

Inhibition of phosphorylation of the oxysterol binding protein by brefeldin A.

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol that localizes to a Golgi/vesicular compartment, migrated on SDS-PAGE as a doublet of 96 and 101 kDa. The reduced mobility of the upper band of this doublet is the result of phosphorylation on multiple serine residues. Phosphorylation of rabbit OSBP stably overexpressed in CHO-K1 cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors such as TPA, sphingosine and bis-indolylmaleimide were without affect. Treatment of overexpressing and control cells with brefeldin A (BFA) caused dephosphorylation of OSBP that coincided with disruption of the Golgi apparatus. [32P]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of dephosphorylation. Phosphopeptide maps of OSBP from overexpressing and control CHO-K1 cells were similar, and BFA promoted dephosphorylation of all five peptides. Compared to overexpressing cells, one tryptic phosphopeptide was more abundant in control CHO-K1 cells and was preferentially dephosphorylated by BFA treatment. OSBP was phosphorylated in vitro by the Golgi enriched fraction of CHO-K1 cells or rat liver by a staurosporine- and BFA-insensitive kinase. The phosphorylation status of OSBP was not affected by 25-hydroxycholesterol and did not alter in vitro 25-[3H]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphorylation of OSBP requires interaction with the Golgi apparatus and an associated kinase. (c) 1998 Elsevier Science B.V.

Involvement of phospholipase D and protein kinase C in phorbol ester and fatty acid stimulated turnover of phosphatidylcholine and phosphatidylethanolamine in neural cells.

Hydrolysis of phosphatidylcholine (PtdCho) can provide lipid second messengers involved in sustained signal transduction. Four neural-derived cell lines (C6 rat glioma; N1E-115 mouse and SK-N-MC and SK-N-SH human neuroblastoma) express different protein kinase C (PKC) isoforms and differentially respond to 4beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA)-stimulation of PtdCho synthesis. We examined involvement of PLD and PKC in the hydrolysis and resynthesis of PtdCho and phosphatidylethanolamine stimulated by beta-TPA, bryostatin (a non-phorbol PKC activator) and oleic acid (18:1n-9) in the four cell lines. beta-TPA or bryostatin produced similar enhancement of [3H]Cho incorporation, loss of stimulated synthesis after down regulation of PKC, and activation of PLD. In C6 cells, staurosporine (STS) and bis-indolylmaleimide (BIM) only partially inhibited basal and beta-TPA-stimulated PLD activity measured as choline or ethanolamine release; phosphatidylbutanol formation after prelabeling with [9,10-3H]18:1n-9, [9,10-3H]myristic acid (14:0), [1-14C]eicosapentaenoic acid (20:5n-3) or 1-O-[alkyl-1', 2-3H]-sn-glyceryl-3-phosphorylcholine gave similar results. STS at >200 nM activated PLD in the presence or absence of beta-TPA. In SK-N-SH cells where PtdCho synthesis was stimulated by beta-TPA or bryostatin, no effect of these agents on PLD was observed. 18:1n-9 stimulated PtdCho synthesis and, to a lesser extent, hydrolysis by PLD both with and without beta-TPA present. Fatty acids had no effect on PKC activities and down regulation of PKC with beta-TPA enhanced fatty acid stimulation of PtdCho synthesis. Thus, activation of PLD hydrolysis preceding resynthesis is involved in the stimulatory effects of beta-TPA on PtdCho synthesis in some but not all of these neural derived cells. Further, PLD hydrolysis of PtdCho and PtdEtn appear to have differing aspects of regulation. Fatty acid regulation of PtdCho synthesis occurs independent of PKC activation. Accordingly, regulation of membrane phospholipid degradation and resynthesis in association with lipid second messenger generation can involve a complex interplay of PLD, PKC, and fatty acids. (c) 1998 Elsevier Science B.V.

Phospholipase D activity is altered in X-linked adrenoleukodystrophy heterozygous carriers, but not in hemizygous patients.

Abnormalities in levels of choline and its metabolites have been reported in the lesions of brains of X-linked adrenoleukodystrophy (X-ALD) patients. We have examined the turnover of the major choline-containing phospholipid, phosphatidylcholine (PtdCho), in fibroblasts from hemizygous X-ALD, heterozygous X-ALD, Zellweger syndrome (ZW), and male and female control individuals to assess possible alterations in PtdCho metabolism mediated by activation of protein kinase C (PKC). Hydrolysis of PtdCho by phospholipase D (PLD) and resynthesis of PtdCho from labeled choline were stimulated 2- to 4-fold by PKC activation with the phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA), in all cells except those from heterozygous X-ALD individuals. No differences in quantity or intracellular distribution of PKC activity, PKC isoforms by Western blot analysis, or of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), were apparent in any of the cells. Thus, altered PtdCho metabolism was not directly linked to either of these inherited defects that result in abnormal peroxisomal functions. Further, altered responsiveness of PLD in X-ALD heterozygotes was independent of changes in PKC and MARCKS.

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells.

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.

Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane.

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells.

Previous studies on neuroblastoma cells in culture showed that the presence of partially purified rat liver phospholipid-transfer protein had a marked differential effect on the uptake and apparent subcellular distribution of radioactively labeled sphingomyelin and phosphatidylcholine (PC) added to the medium as mixed phospholipid (PC/sphingomyelin) liposomes. To determine the effect of phospholipid-transfer protein and exogenous phospholipids on the turnover and subcellular distribution of endogenous phospholipids, neuroblastoma cells were preincubated for 48 h in the presence of [methyl-3H]choline and washed. Aliquots of prelabeled cells were reincubated immediately in medium containing phospholipid-transfer protein mixed phospholipid liposomes, cytochalasin B and 2-deoxyglucose for 45 min at 37 degrees C; additional aliquots were chased first for 2 or 18 h with unlabeled choline before reincubation. The extent of labeled phospholipid degradation and accumulation in the medium, and the subcellular distribution of cell-associated labeled choline-containing phospholipids were determined. During incubation with phospholipid-transfer protein and mixed phospholipid liposomes, 25-35% of the cell-associated radioactive label from prelabeled cells, chased or unchased, was lost to the medium in 45 min. Over 50% of the label appearing in the medium was in water-soluble phospholipid degradation products. The loss of cell-associated label into the medium from unchased cells was stimulated significantly by phospholipid-transfer protein; however, prelabeled cells which had been chased for 18 h with unlabeled choline were unaffected by the presence of transfer protein. Endogenously synthesized radioactively labeled PC and sphingomyelin were distributed throughout all subcellular membranes, but least of all in the crude mitochondrial membrane fraction. Analysis of the subcellular distribution of cell-associated label remaining in chased or unchased cells after 45 min incubation with PC/sphingomyelin liposomes showed proportionate losses from all membrane fractions, except the crude mitochondrial fraction, which showed relative retention of labeled phospholipid. Phospholipid-transfer protein had no effect. The results are in distinct contrast to observations on the turnover, metabolism and subcellular distribution of labeled exogenous phospholipids under the same conditions, indicating that exogenous phospholipids do not intermix freely with any quantitatively major pool of endogenous phospholipid.