PHOSPHOLIPID METABOLISM IN CULTURED NEUROBLASTOMA AND GLIOMA CELLS INCUBATED WITH CARBAMYLCHOLINE AND NOREPINEPHRINE

Cultures of cloned neuroblastoma cells (N1E) in stationary phase and cloned glioma cells (C2₁) in confluency showed substantial differences in phospholipid composition. As a percentage of lipid P, N1E contained more phosphatidylcholine, less ethanolamine phosphoglycerides and much less sphingomyelin than C2₁. When incubated with ³²P_i both cell lines incorporated comparable amounts of radioactivity into total phospholipids. In NIE, phosphatidylcholine contained much more and phosphatidylinositol and phosphatidic acid somewhat less label as compared to C2₁. The presence in the incubation medium of either norepinephrine or carbamylcholine failed to elicit stimulation of ³²P incorporation into any phospholipid class.     

Effect of acute thioacetamide administration on rat brain phospholipid metabolism

Brain phospholipid composition and the [³²P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.     

Effect of freezing and cold storage on phospholipids in developing soybean cotyledons

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. “Harosoy 63” were frozen with liquid N₂, dry ice, or stored in a freezer (−20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at −20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with ³H, and fatty acids with ¹⁴C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.     

Quantitative determination of phospholipids using the dyes Victoria blue R and B

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.     

Saponins can cause the agglutination of phospholipid vesicles

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an α-l-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4–0.5 by a precipitation assay using ¹⁴C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl α-l-arabinofuranoside but not by p-nitrophenyl β-d-glucopyranoside or arabinogalactan. The results indicated that the α-l-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.     

Effects of norepinephrine and acetylcholine on 32P incorporation into phospholipids of the rabbit iris muscle following unilateral superior cervical ganglionectomy.

The effect of norepinephrine and acetylcholine on the ³²P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of ³²P into the phospholipids of the excised iris muscle. $\text{In vivo}$ thr iris muscle incorporated ³²P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while $\text{in vitro}$ phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when ³²Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the ³²P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated ³²P incorporation into phosphatidic acid appears to be post-synaptic.     

Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons

The incorporation of phospholipids specifically labeled with glycerol-2³H and acyl-¹⁴C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-³H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of ³H into triglyceride and therefore N-acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation.     

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.     

A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS–microsome fluorescence. Quantum yield of ANS–microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS–microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS–phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS–microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.     

Effect of hypoglycemia on the brain free fatty acid level and the uptake of fatty acids by phospholipids

The effect of hypoglycemia on the uptake of [1-¹⁴C]arachidonate and [1-¹⁴C]oleate into a synaptosomal and microsomal glycerophospholipids was investigated. In the presence of ATP, Mg²⁺ and CoA, rat brain synaptosomes and micorsomes catalyze the transfer of arachidonate and oleatc into glycerophospholipids. Arachidonate was mainly incorporated into phosphatidylinositol (PI) and phosphatidylcholine (PC), whereas oleate was incorporated into phosphatidylcholine and phosphatidylethanolamine (PE).Hypoglycemia was produced by intraperitoneal injection of 10 or 100 units of crystalline insulin per kg body weight. Two hours after injection the blood glucose level decreased to 10–20 mg%. The content of brain phospholipids was slightly decreased but the change was not statistically significant. The level of free fatty acids (FFA) was increased. More pronounced and reproducible changes were found when hypoglycemia was produced by injection of 100 units of insulin per/kg body weight. Changes in brain cortex were similar to those observed in microsomes and synaptosomes. Hypoglycemia affected the incorporation of arachidonic acid into glycerophospholipids of brain membranes. Uptake of [1-¹⁴C]arachidonate was decreased selectively by 50% (into phosphatidic acid /PA/) when hypogiycemia was produced by injection of 10 units of insulin per kg body weight. The Higher dose of insulin 100 units per kg body weight produced a 20% inhibition of arachidonate incorporation into synaptosomal PI and a 13% decrease of incorporation into microsomal phosphatidylcholine. Incorporation of [1-¹⁴C]oleate into membrane phospholipids was not changed by hypoglycemic insult. It is proposed that the disturbances in fatty acid level, particularly arachidonate, and decreased uptake of arachidonic acid by synaptosomal glycerophospholipids may be responsible for alteration of membrane function and changes of synaptic processes.     

Insulin inhibits changes in the phospholipid profiles in sciatic nerves from streptozocin-induced diabetic rats: A phosphorus-31 magnetic resonance study

Sciatic nerve phospholipids obtained from insulin-treated streptozocin-induced diabetic, non-treated streptozocin-induced diabetic, and healthy, control male Sprague-Dawley rats after eighteen weeks of diabetes were studied by ³¹P NMR spectrometry. Eleven phospholipids resonances were identified as follows: Phosphatidic acid (Chemical shift, 0.30 ppm), dihydrosphingomyelin (0.13 ppm), ethanolamine plasmalogen (0.07 ppm), phosphatidylethanolamine (0.03 ppm), phosphatidylserine (−0.05 ppm), sphingomyelin (−0.09 ppm), lysophosphatidylcholine (−0.28 ppm), phosphatidylinositol (−0.30 ppm), alkylacylglycerophosphorylcholine (−0.78 ppm), choline plasmalogen (−0.80 ppm), and phosphatidylcholine (−0.84 ppm). Diabetic rats showed that phosphatidylcholine was significantly elevated p > 0.05, and ethanolamine plasmalogen and choline plasmalogen were significantly lower when compared with both control and insulin treated rats. The choline ratio (choline-containing phospholipids over noncholine phospholipids) was significantly elevated in the diabetic group, when compared with both control and insulin-treated groups. The ethanolamine ratio (ethanolamine-containing phospholipids over nonethanolamine phospholipids) and the ratio of the ethanolamine ratio over the choline ratio, was significantly elevated in the control and the insulin-treated groups when compared with the diabetic group. The presence of phosphatidic acid and the significance in phosphatidylcholine and ethanolamine plasmalogen, suggested that insulin had a role in the phosphatidylcholine metabolism in the rat nerve.     

Interaction of antibody-bearing small unilamellar liposomes with antigencoated cells. The effect of antibody and antigen concentration on the liposomal and cell surface respectively

Human blood lymphocytes were coated with increasing amounts of human kappa chain (2–85μg/10⁷ cells) through the linking reagent CrCl₃. These cells were then exposed to small unilamellar liposomes composed of egg phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 7:7:1) containing carboxyfluorescein and/or ¹¹¹In-labelled bleomycin and bearing ¹³¹I-labelled affinity chromatography-purified or non-purified anti-(kappa-chain) immunoglobulin G (IgG) [see the preceding paper, Gregoriadis, Meehan & Mah (1981) Biochem. J. 200, 203–210]. In some experiments liposomes contained [¹⁴C]phosphatidylcholine. (1) Lymphocytes (10⁷) coated with 2–85μg of kappa chain and exposed to liposomes devoid of IgG or bearing non-purified anti-(kappa chain) IgG bound only a small proportion of the liposomal markers. Even with liposomes bearing the purified anti-(kappa chain) IgG, uptake of the labels improved only slightly for cells coated with up to 10μg of kappa chain. However, with higher concentrations of the antigen on the cell surface, binding was improved considerably to reach values of 31% (¹¹¹In-labelled bleomycin) and 43% (¹³¹I-labelled IgG) of added liposomes for cells coated with 85μg of kappa chain. (2) Lymphocytes coated with kappa chain were exposed to liposomes bearing increasing amounts (0–180μg/0.9mg of egg phosphatidylcholine) of purified anti-(kappa chain) IgG. It was found that under the present conditions, binding of all three markers (¹¹¹In-labelled bleomycin, ¹³¹I-labelled IgG and [¹⁴C]phosphatidylcholine) was directly proportional to the concentration of IgG on the liposomal surface. However, uptake values remained unchanged above 90μg of IgG. (3) Antibody-mediated uptake of liposomes by cells coated with the corresponding antigen without loss of their metabolic activities may provide a method of efficient targeting.     

Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [³²P]P_i into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [³²P]P_i into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [³²P]P_i into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.     

Insulin stimulates turnover of phosphatidylcholine in rat adipocytes

The present study investigated the effect of insulin on phosphatidylcholine turnover in rat adipocytes labelled to equilibrium with [¹⁴C]-choline. Insulin induced a rapid turnover of this major phospholipid that was maximal by 1 min and transient in nature. Following a 1 min stimulation of the cells with insulin at a maximally effective concentration (7 nM), a 4–6% decrease in the percentage of total cellular choline associated with this phospholipid was observed. This reflected a significant transient increase in the percentage of total cellular choline associated with phosphorylcholine, which together with diacylglycerol are the phospholipase C cleavage products of phosphatidylcholine. These effects were observed over a physiological range of insulin concentrations. No effect of insulin on any other choline phospholipid or metabolite (sphingomyelin, lysophophatidylcholine, glycerophosphocholine or choline) was seen. These results suggest that insulin stimulates a phospholipase C-mediated turnover of phosphatidylcholine in rat adipocytes. The rapid nature of this turnover suggests a potential role in signal transduction.     

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine.     

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

(1) The hydrolysis of ³²P- or myo-[2-³H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca²⁺ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca²⁺ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[³H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.     

Lipophorin from Rhodnius prolixus: Purification and partial characterization

The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with ³²P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [³²P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction ³²P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.     

The phospholipid requirement of the (Ca + Mg)-ATPase from human platelets

The phospholipid requirement of the (Ca²⁺ + Mg²⁺)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.     

The role of charge in lipid selectivity for the nicotinic acetylcholine receptor.

The effect of salt and pH titration on the selectivity of spin-labeled analogues of phosphatidic acid, phosphatidylserine, phosphatidylcholine, and stearic acid for the nicotinic acetylcholine receptor (nAcChoR) reconstituted into dioleoylphosphatidylcholine was examined at 0 degrees C using electron spin resonance spectroscopy. The order of selectivity at pH 7.4 and 0 mM NaCl was phosphatidylserine > stearic acid > phosphatidic acid > phosphatidylcholine. The addition up to 2 M NaCl or titration of pH from 5.0 to > 9.0 did not alter the selectivity of the phospholipids for the nAcChoR. For stearic acid, conversely, titration of pH from 5.0 to 9.0 at 0 mM NaCl and titration of NaCl from 0 to 2 M at pH 9.0 both increased selectivity for the nAcChoR. It is concluded that electrostatic interactions do not account for the selectivity of the negatively charged phospholipids, phosphatidylserine, and phosphatidic acid for the nAcChoR. This is consistent with the known orientation of the transmembrane sequences M1 and M4, which predicts a balance in the number of negative and positive charges in the lipid-protein interface and suggests that the two positive charges on each M3 helix are not exposed to the lipid-protein interface.