Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.     

Molecular species analysis of a product of phospholipase D activation. Phosphatidylethanol is formed from phosphatidylcholine in phorbol ester- and bradykinin-stimulated PC12 cells.

Tumor-promoting phorbol esters or calcium-mobilizing receptor ligands stimulate phosphatidylcholine breakdown and in many cells this is accompanied by phospholipase D (PLD) activation. We tested whether or not a direct relationship exists between these two phenomena. Pheochromocytoma (PC12) cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate or with the calcium-mobilizing receptor ligand bradykinin in media containing 1% ethanol. The fatty acid composition of the molecular species of phosphatidylethanol (PEt), a product of PLD activation, formed in stimulated cells was compared with the molecular species of endogenous phospholipids isolated from unstimulated PC12 cells. PEt was isolated and analyzed by fast atom bombardment-mass spectrometry (FAB-MS) in the negative ion mode. Fatty acid composition and headgroup structure of the major PEt molecular ions were confirmed by linked scan analysis. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were isolated from unstimulated cells and converted into phosphatidic acids using PLD. Mass spectra of the respective phosphatidic acids were obtained by fast atom bombardment-mass spectrometry as described above. The molecular species of PEt formed in 12-O-tetradecanoylphorbol-13-acetate- and bradykinin-stimulated PC12 cell were identical to those of phosphatidylcholine isolated from untreated cells.     

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Decreased Membrane Integrity in Aging Typha latifolia L.Pollen (Accumulation of Lysolipids and Free Fatty Acids)

Aging of cattail (Typha latifolia L.) pollen was studied at 24[deg]C under conditions of 40 and 75% relative humidity (RH). The decline of viability coincides with increased leakage at imbibition; both processes develop much faster at the higher humidity condition. During aging phospholipids are deesterified and free fatty acids (FFAs) and lysophospholipids (LPLs) accumulate, again, much more rapidly at 75% RH than at 40% RH. The fatty acid composition of the remaining phospholipids hardly changes during aging, which suggests limited involvement of lipid peroxidation in the degradation process. Tests with phospholipase A2 revealed that the saturated fatty acids occur at the sn-1 position of the glycerol backbone of the phospholipids. The fatty acid composition of the LPLs is similar to that of the phospholipids from which they were formed, indicating that the deesterification occurs at random. This favors involvement of free radicals instead of phospholipases in the deesterification process. Liposome studies were carried out to characterize components in the lipid fraction that might account for the leakage associated with aging. Entrapped carboxyfluorescein leaked much more from liposomes when they were partly made up from total lipids from aged pollen than from nonaged pollen. The components causing the leakage were found in both the polar and the neutral lipid fractions. Further purification and subsequent interchanging of the FFAs and LPLs between extracts from aged and nonaged pollen revealed that in neutral lipid extracts the FFAs are entirely responsible for the leakage, whereas in the phospholipid fraction the LPLs are largely responsible for the leakage. The leakage from the liposomes is not caused by fusion. We suggest that the observed loss of viability and increased leakage during aging are due to the nonenzymic accumulation of FFAs and LPLs in the pollen membranes.     

Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

The effects of mastoparan on the carrot cell plasma membrane polyphosphoinositide phospholipase C.

When [3H]inositol-labeled carrot (Daucus carota L.) cells were treated with 10 or 25 microM wasp venom peptide mastoparan or the active analog Mas-7 there was a rapid loss of more than 70% of [3H]phosphatidylinositol-4-monophosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) and a 3- and 4-fold increase in [3H]inositol-1,4-P2 and [3H]inositol-1,4,5-P3, respectively. The identity of [3H]inositol-1,4,5-P3 was confirmed by phosphorylation with inositol-1,4,5-P3 3-kinase and co-migration with inositol-1,3,4,5-P4. The changes in phosphoinositides were evident within 1 min. The loss of [3H]PIP was evident only when cells were treated with the higher concentrations (10 and 25 microM) of mastoparan or Mas-7. At 1 microM Mas-7, [3H]PIP increased. The inactive mastoparan analog Mas-17 had little or no effect on [3H]PIP or [3H]PIP2 hydrolysis in vivo. Neomycin (100 microM) inhibited the uptake of Mas-7 and thereby inhibited the Mas-7-stimulated hydrolysis of [3H]PIP and [3H]PIP2. Plasma membranes isolated from mastoparan-treated cells had increased PIP-phospholipase C (PLC) activity. However, when Mas-7 was added to isolated plasma membranes from control cells, it had no effect on PIP-PLC activity at low concentrations and inhibited PIP-PLC at concentrations greater than 10 microM. In addition, guanosine-5'-O-(3-thiotriphosphate) had no effect on the PIP-PLC activity when added to plasma membranes isolated from either the Mas-7-treated or control cells. The fact that Mas-7 did not stimulate PIP-PLC activity in vitro indicated that the Mas-7-induced increase in PIP-PLC in vivo required a factor that was lost from the membrane during isolation.     

Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids

Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH.     

N-3 polyunsaturated fatty acid supplementation alters inositol phosphate metabolism and protein kinase C activity in adult porcine cardiac myocytes

Several mechanisms have been proposed to explain the anti-arrhythmic effects of n-3 polyunsaturated fatty acids. One mechanism is the effect of modifying cell membrane phospholipid and their subsequent effect on intracellular cell signaling via the second messengers, Ins(1,4,5)P(3) and diacylglycerol. Isolated cardiac myocytes from adult pig hearts were used to investigate the effect of n-3 polyunsaturated fatty acids, eicosapentaenoic acid and docosahexaenoic acid, on the inositol phosphate metabolism and protein kinase C activity. Adult porcine cardiac myocytes were grown in media supplemented with 400 μM arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. After 24 hr, fatty acid analyses of total lipids by TLC in supplemented cells showed that eicosapentaenoic acid and docosahexaenoic acid were selectively incorporated into the phosphatidylinositol fraction. In the diacylglycerol fraction, there was a small incorporation of both eicosapentaenoic acid and docosahexaenoic acid but it was not significantly different from that of controls. To study the effect of membrane phospholipid modification on the phospholipase C mediated inositol lipid cycle, cardiac myocytes were labeled with 4μCi/ml myo-[2-(3)H]Ins for 48 hr. After stimulation with epinephrine and phenylephrine (alpha-receptor agonist) the water soluble [(3)H]Ins products were separated by chromatography on Dowex AG 1-X8 and measured by scintillation counting. After stimulation, the levels of [(3)H]Ins(1,4,5)P(3) and [(3)H]Ins(1,3,4,5)P(4) in eicosapentaenoic acid and docosahexaenoic acid supplemented myocytes were significantly reduced (P < 0.05) compared to arachidonic acid supplemented myocytes. Similarly, eicosapentaenoic acid and docosahexaenoic acid supplemented cells had reduced levels of protein kinase C activity after stimulation compared to arachidonic acid supplemented cells. From these experiments, it is evident that n-3 PUFA supplementation modulates intracellular cell signaling suggesting a possible anti-arrhythmic mechanism.     

Adriamycin transport and sensitivity in fatty acid-modified leukemia cells

The membrane phospholipids of L1210 murine leukemia cells were modified by supplementing the growth medium with micromolar concentrations of polyunsaturated or monounsaturated fatty acids. This procedure results in enrichment of cellular phospholipids by the supplemented fatty acid. Enrichment with polyunsaturated fatty acids resulted in a marked increase in sensitivity to adriamycin as compared to enrichment with monounsaturated fatty acids. The increased cytotoxicity was directly proportional to the extent of unsaturation of the inserted fatty acid, but there was no difference in cells enriched with n-3 compared with n-6 family fatty acids. To explore the mechanism of this observation, we examined whether augmented uptake of the drug might explain the increased cytotoxicity. The uptake of [14C]adriamycin, which was approximately linear at later time points, was only partially temperature dependent and never reached a steady state. Initial uptake at time points prior to 60 s could not be measured due to high and variable rapid membrane adsorption. Cellular accumulation of drug was greater in the docosahexaenoate 22:6-enriched L1210 cells as compared to oleate 18:1-enriched cells and was about 32% greater after 20 min. When L1210 cells were enriched with six fatty acids of variable degrees of unsaturation, the accumulation of adriamycin was directly correlated with the average number of double bonds in the fatty acids contained in cellular phospholipids. There was no difference in efflux of drug from cells pre-loaded with adriamycin. We conclude that the greater accumulation of adriamycin by the polyunsaturated fatty acid-enriched L1210 cells likely explains the increased sensitivity of these cells to adriamycin compared to 18:1-enriched cells.     

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): Detection of a high level of arachidonic acid

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C₂₀ polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A₂ showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A₂ for subsequent metabolism to prostaglandins and/or other eicosanoids. © 1993 Wiley-Liss, Inc.     

Lipid metabolism in human endothelial cells

Although lipids are largely involved in cardiovascular physiopathology, the lipid metabolism in endothelial cells remains largely unknown. Human umbilical vein endothelial cells (HUVECs) were used to investigate the metabolism of complex lipids. The membrane phospholipid homeostasis results from both de novo synthesis and remodelling that ensures the fine tuning of the phospholipid fatty acid composition. Using [(3)H]-glycerol and phosphoderivatives we showed the efficiency of glycerolipid synthesis from glycerol (0.9 nmol h(-1) mg proteins(-1)), but not from its phosphorylated form suggesting the requirement of a functional glycerol kinase in HUVECs. Conversely, the synthesis of triacylglycerols was very low (less than 5% of phospholipid synthesis). The incorporation rate of fatty acids into phospholipids showed that there is a specific fate for each fatty acid in respect to its chain length and saturation level. Moreover in steady state condition, increasing the long chain omega3 polyunsaturated fatty acids in the medium resulted in an increased polyunsaturated/saturated ratio in phospholipids (from 0.42 to 0.63). [(14)C]O(2) was produced form either [(14)C]-glucose or [(14)C]-palmitate indicating the functionality of the oxidation pathways, although beta-oxidation was less efficient than glucose oxidation. The endothelial cell lipid metabolism involves conventional pathways, with functional rates largely slower than in hepatocytes or in cardiomyocytes.     

Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.     

n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4(+) T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Ctheta, phospholipase Cgamma-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cgamma-1 at the IS and cell proliferation as assessed by CFSE labeling and [(3)H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.     

The relationship between plasma membrane lipid composition and physical-chemical properties. III. Detailed physical and biochemical analysis of fatty acid-substituted EL4 plasma membranes

Murine leukemia EL4 cells were modified by supplementation of culture media with fatty acids for 24 h. A plasma membrane-enriched fraction was prepared from substituted and normal cells. Analyses were performed to determine fatty acyl composition, phospholipid headgroup composition and cholesterol content. The two major membrane phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were isolated by thin-layer chromatography and ESR measurements were done on liposomes prepared from these lipids as well as on the intact plasma membrane preparations. Slight perturbations in overall plasma membrane lipid composition were observed when EL4 cells were supplemented with a single exogenous fatty acid. This may be consistent with the idea that the incorporation of exogenous fatty acid induces compensatory changes in membrane lipid composition. On the other hand, we observed no significant difference in two ESR motional parameters between the unsubstituted control and various fatty acid-substituted plasma membranes. ESR measurements carried out on PE and PC liposomes derived from 17:0- and 18:2c-substituted membranes also failed to detect major differences between these liposomes and those made from normal EL4 phospholipids. In the case of liposomes prepared from 18:2t,-substituted membranes, the order parameter was significantly changed from the normal. However, the change was in opposite directions in PE and PC, perhaps accounting for the fact that no change parameter is seen in intact 18:2t-substituted plasma membrane. Measurements of order parameter (S) in mixed lipid vesicles showed that at up to 50 mol% mixture of a synthetic PC with plasma membrane PC, the value of S was only marginally different from that of the plasma membrane PC vesicles. We interpret these data as an indication that the two ESR parameters used are not sufficiently sensitive to detect changes due to modifications of the acyl chain composition of a complex biological membrane.     

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.     

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophyte's tactics for salt tolerance.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.     

Phospholipase D in homogenates from HL-60 granulocytes: implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.