Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [³²P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine to the medium,¹⁴C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,¹⁴C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the¹⁴C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min. Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A₁ and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A₂ was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A₁ and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.     

Regulation of phosphatidylinositol turnover,calcium metabolism and enzyme secretion by phorbol dibutyrate in neutrophils

The action of the tumor promoter, phorbol 12,13-dibutyrate (PDBu), on rabbit peritoneal and human neutrophils is associated with stimulation of¹⁴C-arachidonic acid incorporation into phospholipids within 1–2 min. Stimulated¹⁴C-arachidonate incorporation was relatively selective for phosphatidylinositol (PI) in rabbit neutrophils. In contrast, the secretory response of human neutrophils to PDBu coincided with stimulated label incorporation into phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and PI. Significant increases in label incorporation were observed with PDBu concentrations as low as 2 nM, and the dose response of stimulated label incorporation paralleled that of evoked lysozyme secretion. A parallel, but partial, inhibition of PDBu-stimulated PI labeling and enzyme release was observed after exposing rabbit neutrophils to calcium-deprived medium, whereas calcium deprivation failed to significantly depress either of these stimulant actions of PDBu in human neutrophils. Further, in rabbit neutrophils PDBu elicited an increase in cell associated⁴⁵Ca. However, PDBu was unable to promote the incorporation of³²P orthophosphate into PI or enhance phospholipase A₂ activity in broken cells. These findings suggest that one expression of the interaction between phorbol esters and their receptors on neutrophils involves the turnover of arachidonic acid in phospholipids. This stimulated turnover of arachidonate may be a critical step in the cascade of events associated with neutrophil activation.     

Metabolism of labeled isomeric octadecenoates by the laying hen

Discrimination between octadecenoic acid isomers by the laying hen has been studied using tritium (³H), carbon-14 (¹⁴C) and deuterium (d) labeled oleate and elaidate esters. Hydrogen isotopes were positioned at the double bond, whereas¹⁴C was located in the 1-, carboxyl carbon. The egg acted as a biological trap, providing an automatic daily biopsy with which to study the metabolism of the fed isomers. Monitoring the incorporation of isomers was facilitated by dual label feeding experiments, and³H/¹⁴C, d₂/d_o and d₂/d₁ ratios were determined on the isomeric mixtures fed, on the total egg lipids extracted and on the isolated neutral lipid and phospholipid fractions. Comparison of isotopic ratios of the fed mixture and of the lipid fractions provided an evaluation of discrimination by the hen during the transport of isomeric octadecenoates into the egg lipids. Radioactive and stabl isotope ratios determined for the neutral lipid indicated a preferential incorporation of thecis isomer. Stable isotope ratios determined for the phospholipid showed that thetrans isomer is preferentially incorporated. The³H/¹⁴C ratios for the phospholipid recovered in each experiment increased greatly whichever isomer was labeled with³H, indicating an elimination of the 1-¹⁴C-label. Gas liquid radiochromatographic separation of the methyl esters from the neutral lipids and phospholipids showed that the isotopic labels were present almost exclusively in the octadecenoic acid constituent. Presented at the AOCS Meeting, Houston, May 1971. No. Mktg. and. Nutr. Res. Div., ARS, USDA.     

Erucic acid and phospholipids of newborn rat heart cells in culture

Erucic acid (Δ¹³-docosenoic acid), labeled with¹⁴C in the 1-or 14-position, was incorporated into fetal calf serum and fed to beating, neonatal rat myocardial cells in culture. Uptake of the docosenoic acid during the first 6 hr of incubation was 41 nM/hr/mg protein in 7-day old cells and 29 nM/hr/mg protein in 14-day old cells. Fifty-seven percent of the¹⁴C-activity was taken up from the medium in 24 hr, of which 77% was in the cells and 23% was unaccounted for. Of the¹⁴C-activity taken up, 26% was in extractable lipid, with two-thirds in neutral lipid and one-third in phospholipid. Within the neutral lipid fraction, 88% of the¹⁴C-activity was present in triglycerides; while in phospholipids, 66% of the¹⁴C-activity was in phosphatidylcholine (PC); 14% in phosphatidylethanolamine (PE); 6% in sphingomyelin (SPH) and 1% or less in cardiolipin (DPG). PC had the highest specific activity, followed by SPH and PE. The specific activity of PE was one-half that of SPH when the¹⁴C-erucic acid substrate was labeled at the carboxyl position, but increased to equal that of SPH when the substrate was labeled at the double bond. The fatty acids of PC, PE, and SPH were influenced by erucic acid in the growth medium, but the amounts of each phospholipid were not affected. It is proposed that the altered fatty acid composition associated with incorporation of erucic acid or its metabolites into PC, PE, and SPH may affect integrity and function of heart cell membranes.     

Phospholipid studies of marine organisms: New branched fatty acids fromStrongylophora durissima

The phospholipids of the spongeStrongylophora durissima were analyzed. The major phospholipids present were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). The major fatty acid components of the phospholipids consisted of short chain (C₁₄−C₁₉) and very long chain (C₂₅−C₃₀) “Demospongic” acids. Three novel branched Δ⁵ monounsaturated acids,Z-19-methyl-5-pentacosenoic,Z-19-methyl-5-hexacosenoic andZ-19-methyl-5-heptacosenoic acids were encountered in the sponge. The 3-saturated counterparts of these compounds, 19-methylpentacosanoic, 19-methylhexacosanoic and 19-methylheptacosanoic acids, as well as 19-methylpentacosanoic and 20-methyloctacosanoic acids also are hitherto undescribed acids present in the sponge. Trace amounts of 2 very long chain acids also were detected and their structures tentatively assigned as 19,21-dimethylheptacosanoic and 20,22-dimethyloctacosanoic acids. The distribution of these fatty acids according to phospholipid head groups also was described.     

Phospholipid exchange between subcellular organelles of rabbit lung

A soluble protein fraction (PLEP) prepared from rabbit lung can catalyze the exchange of phospholipids between subcellular organelles of the lung and between these subcellular organelles and synthetic liposomes. Phospholipid exchange between microsomes and synthetic liposomes and between mitochondria and synthetic liposomes was stimulated 8-fold and 2.5-fold, respectively, in the presence of the protein fraction. Lung exchange protein could also catalyze phospholipid exchange between subcellular organelles of the liver and synthetic liposomes. Phospholipid transfer between microsomes and lamellar bodies of the lung was stimulated 2-fold by the exchange protein. Both radiolabeled phosphatidylcholine (PC) and phosphatidylinositol (PI) were transferred from³²P-labeled microsomes to lamellar bodies, but the exchange protein exhibited no transfer activity for phosphatidylglycerol (PG) and that for phosphatidylethanolamine (PE) was insignificant compared to the transfer activity for phosphatidylcholine and phosphatidylinositol. While the physiological role of the phospholipid exchange proteins in the lung is unknown, it is possible that they participate in the distribution of the newly synthesized phospholipids from the site of synthesis to lamellar bodies and other membrane compartments of cells.     

Lipid composition of subcellular membranes from larvae and prepupae ofDrosophila melanogaster

Subcellular membranes were analyzed for their lipid composition and protein content at two developmental points representing the third instar wandering larvae and prepupal stages ofDrosophila. At both stages, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major constituents with phosphatidylinositol (PI), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) being relatively minor components. In total homogenates and in the nuclear-enriched fraction there was no significant difference in the phospholipid composition of the wandering larvae and prepupae. In mitochondria only a significant increase in the minor component PS was observed in the prepupae. In lysosomal membranes on the other hand, the relative abundance of the major components PE and PC increased in the prepupae although the molar ratios of the two lipids remained almost constant. The fatty acid composition of the phospholipids remained virtually unchanged in all of the fractions examined, including the lysosomes, and there was no evidence of lipid peroxidation. With regard to cellular degeneration and the involvement of lysosomes, we conclude that mechanisms other than gross modification of the lipid and/or lipid/protein ratio of their membranes are involved in the liberation of the acid phosphatase contents.     

Meningioma phospholipid profiles measured by31P nuclear magnetic resonance spectroscopy

Fourteen cases of intracranial meningioma were characterized after chloroform/methanol extraction by³¹P nuclear magnetic resonance (NMR) spectroscopy at 202.4 MHz. Each phospholipid class detected in the extracts was identified and quantitated in terms of its molar percentage relative to the total phospholipids measured. The following phospholipids were assayed by³¹P NMR: phosphatidylglycerol, phosphatidic acid, diphosphatidylglycerol, ethanolamine plasmalogen, phosphatidylethanolamine (PE), lysophosphatidylinositol, phosphatidylserine, sphingomyelin, lysophosphatidylcholine (LPC), phosphatidylinositol (PI), sphingosylphosphorylcholine and phosphatidylcholine. In addition, two unidentified phospholipids were detected with resonances at 0.13 and −0.78 ppm, respectively. Three distinct types of spectra were obtained on the extracts and grouped accordingly for comparison purposes. Type 1 tumors showed unusual³¹P NMR profiles with low levels of PE and PI and elevated levels of LPC; type 2 tumors were characterized by low levels of the ethanolamine phospholipids and near equivalent levels of PI and LPC. The spectra of type 1 and type 2 tumors were characteristic of degenerative cells that lacked membrane permeability associated with loss of ethanolamine plasmalogen in the presence of significant phospholipid turnover. Meningiomas belonging to the third spectral type showed characteristics similar to those of normal tissues with normal levels of PE and ethanolamine plasmalogen, as well as very low levels of LPC relative to PI. Type 3 tumors lacked the characteristic signs of degeneration noted in type 1 and type 2 tumors. The data corroborate and augmentin vivo spectroscopic findings reported earlier and demonstrate the value of³¹P NMR spectroscopic phospholipid analysis on lipid extracts for the characterization of meningiomas.     

Is there an entero-hepatic circulation of the bile phospholipids?

Bile previously labeled with tritiated oleic acid (the main radioactivity was on bile phospholipids) was mixed with pure isolated phospholipids previously labeled with¹⁴C oleic acid; this mixture was perfused during 6 or 23 hr into the duodenum of test rats bearing a bile fistula. At the time of decapitation, in the small intestine a large hydrolysis of the¹⁴C phospholipids was found. In contrast no bile phospholipid hydrolysis was observed. In the collected bile samples of the test rats, no¹⁴C could be detected (this means a very large decrease of the¹⁴C fatty acids specific activities by the body fatty acids), and the tritiated fatty acids specific activities were only 2.5–12 times lower than in the perfused bile. These results can be explained, assuming that the bile phospholipids enter in an entero-hepatic circulation and are preserved from the dilution in a large pool of lipids.     

Enzymatic method of increasing phosphatidylcholine content of lecithin

An enzymatic method was established to increase the phosphatidylcholine (PC) contents of soybean and egg lecithins. Other phospholipids of lecithin were phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidic acid (PA). Seven preparations of phospholipase D (PLD), PLD-1 to PLD-6 ofStreptomyces origin and PLD-7 of cabbage origin, were tested for their ability to increase PC by transphosphatidylation in the presence of choline chloride (CC). The reactions were carried out at 30 C in a biphasic system that consisted of an aqueous phase containing PLD along with a buffer (optimum pH) having desired concentration of CC and Ca²⁺ and an ethyl acetate phase containing lecithin phospholipids. Intermitttent samples were extracted and analyzed by HPLC. Four of six PLD’s ofStreptomyces origin showed good transphosphatidylation (increase of PC contents of soybean lecithin from approximately 35% to 60–70% on a phospholipid basis) at 2.5 M CC, but the other two microbial PLD’s completely hydrolyzed the phospholipids to PA. Cabbage PLD-7 showed poor transphosphatidylation. PLD-3 gave the highest PC contents (70%) at 1.75 M CC. One hundred percent transphosphatidylation of pure PE to PC was achieved with PLD-3. PI was inert to the attack of most PLD preparations examined with the exception that PLD-3 hydrolyzed PI significantly. Purified PI could not be transphosphatidylized to PC; 100% PA was formed. Soybean lecithin containing about 80% PC and purified egg yolk lecithin with 75% PC could be converted to products having 95% PC and almost 100% PC, respectively, by PLD-3 at 1.75M CC. Studies on Enzymatic Conversion of Phospholipids (v)     

Incorporation of [1-14C] acetate into lipids of soybean cell suspensions

Suspension cultures of soybean cells incorporated [1-¹⁴C] acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 min; more than 75% of this label was found to be in the oleic acid of the phosphatidylcholine. After longer periods of incubation, the proportion of¹⁴C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively,bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglycerols, and the glycolipid fraction. Contribution no. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977.     

Incorporation of lipids labeled with various fatty acids into the cytoskeleton of aggregating platelets

Earlier studies showed that during the first 20 to 25 seconds of aggregation induced by thrombin (0.1 U/mL) or adenosine diphosphate (ADP) (2μM) of rabbit or human platelets prelabeled with [³H]palmitic acid, labeled lipid became associated with the cytoskeleton (isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(β-aminoethyl ether(N,N,N′,N′-tetraacetic acid] in the presence of 0.5 mM leupeptin and 50 mM benzamidine). In comparison with labeled lipid in intact platelets, the labeled lipid that was associated with the cytoskeleton was enriched in phospholipids and ceramide. To determine whether these effects were specific for lipids labeled with palmitic acid, we studied rabbit platelets in which lipids had been labeled by incubation of the platelets with pairs of¹⁴C- or³H-labeled palmitic, stearic, arachidonic, and linoleic acids. Examination of the distribution of label among the lipid classes of intact platelets showed that phospholipids contained most of the label. Under the conditions of limited, thrombin-induced aggregation used, labeled lipids were not lost from the platelets and the distribution of label among the lipid classes was essentially unchanged. There were major differences in the incorporation of labeled lipids into the cytoskeleton. The greatest incorporation (2.1 to 2.8% of the label in the platelets) was observed with palmitic acid-labeled lipids; by direct comparison, only 44% as much of the label of stearic acid-labeled lipids, 21% as much of the label of linoleic acid-labeled lipids, and only 6% as much of the label of arachidonic acid-labeled lipids was incorporated into the cytoskeleton. Thus the pool of phospholipid that is readily labeled with arachidonic acid appears to be selectively excluded from the cytoskeleton. Also noteworthy is the 4- to 5-fold enrichment of the cytoskeleton with labeled ceramide; an average of 16% of the label from stearic acid in the cytoskeleton was in ceramide. We suggest that ceramide and phospholipids that are readily labeled with saturated fatty acids are selectively incorporated into the cytoskeleton during the early stages of aggregation and may be specifically associated with the points of contact between platelets.     

Phospholipids inTrypanosoma cruzi: Phosphoinositide composition and turnover

The polyphosphoinositides fromTrypanosoma cruzi were isolated by preparative thin-layer chromatography (TLC) and identified. When myo-[³H]inositol was present in the culture medium for five days, analyses showed the presence of phosphatidylinositol (PI), lysophosphatidylinositol (lysoPI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂). Short-term incubation with³²P_i led to higher percentages of incorporation into phosphatidylethanolamine (PE), lysophosphatidylethanolamine (lysoPE) and PI compared to the other glycerophospholipids. The phosphoinositides (PI, PIP and PIP₂) contained a larger proportion of unsaturated than saturated fatty acids. High proportions of 18∶2 were found in the three phosphoinositides analyzed, whereas the major saturated fatty acid was 18∶0. Watersoluble inositol phosphates (IP, IP₂ and IP₃) were also identified.     

31P NMR of tissue phospholipids: Competition for Mg2+, Ca2+, Na+ and K+ cations

Phosphatidylcholine (PC), phosphatidylethanolamine (PE), ethanolamine plasmalogen (EPLAS), sphingomyelin (SPH), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidic acid (PA) were dispersed together in Cs(ethylenedinitrilo)tetraacetic acid-scrubbed chloroform/methanol solution, and high resolution³¹P nuclear magnetic resonance spectra were recorded. In separate titration experiments, Mg²⁺ and Ca²⁺ were added to the dispersed phospholipid mixture to determine the relative interaction potentials of each of the phospholipids for each of the added cations. The association of cations with individual phospholipids was indicated by³¹P chemical-shift changes, signal broadening, signal quenching or a combination of these. The titrations revealed that CL had the highest, and PA the next highest, interaction potential for Mg²⁺ cations. In contrast, PS and PA had the highest, and CL the next highest, interaction potential for Ca²⁺. Considering only interactions with Ca²⁺ ions, the phospholipids can be divided into three distinct groups: PS and PA (high interaction potential); CL, PI and PG (intermediate interaction potential); and EPLAS, PE, SPH and PC (essentially no interaction potential). The two phospholipids with the least interaction potential for either of the alkaline-earth cations were PC and SPH. Na⁺ and K⁺ ion interactions with PA, CL, PI and PG were unique and resulted in positive chemical-shift changes relative to the chemical shifts in the presence of Cs⁺ ions. Relative to both Cs⁺ and K⁺ ions, chemical shifts in the presence of Na⁺ ions were deshielded δ>0.1 ppm in the order PA>CL>PI>PG.     

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells,vessel walls and platelets

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with¹⁴C-eicosapentaenoic acid and¹⁴C-arachidonic acid revealed that the incorporation of¹⁴C-eicosapentaenoic acid into the phosphatidylinositol fraction is significantly less than that of¹⁴C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I₃ by the vascular cells nor to thromboxane A₃ by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.     

Platelet-activating factor regulates phospholipid metabolism in human neutrophils

This study extended the earlier finding that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) promotes arachidonic acid incorporation into neutrophil phosphatidylinositol (PI) and phosphatidylcholine (PC). In the present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10⁻⁷ M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1–10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC. These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF.     

Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation

Previous experiments showed that during the early stages (20–30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 μM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [³H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(β-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species and with either ADP or thrombin as aggregating agent, 70–85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton. Although intact rabbit platelets had more label in ceramide (6.7±2.9%) than intact human platelets (1.5±0.9%), platelets of both species exhibited a three- to four-fold enrichment of labeled ceramide in the cytoskeletons. Thus phospholipids and ceramide that are readily labeled with palmitic acid are selectively incorporated into the cytoskeleton during the initial stages of platelet aggregation.     

Arachidonic acid uptake by phospholipids and triacylglycerols of rat brain subcellular membranes

In the presence of ATP, MgCl₂ and CoASH, different subcellular membrane fractions isolated from rat cerebral cortex exhibited characteristic profiles for the incorporation of [1-¹⁴C]arachidonic acid into phospholipids and triacylglycerols. In general, uptake of label by phosphatidylcholines was higher in the synaptic membranes, and that by phosphatidylinositols was higher in the microsomes and somal plasma membranes. A substantial amount of the labeled arachidonate was also incorporated into triacylglycerols, especially in the somal plasma membranes and microsomes. Enzymes mediating the transfer of arachidonic acid to phospholipids were unstable with respect to sample storage and exposure to elevated temperatures. In contrast, the acyltransferase for triacylglycerols was more stable to these factors. Washing the membranes with bovine serum albumin resulted in an enhancement of the incorporation of label into phosphatidylinositols without affecting that of phosphatidylcholines, but the incorporation into triacylglycerols was inhibited. Treatment of synaptosomes and plasma membranes with saponin resulted in an enhancement in the labeling of phospholipids, but the labeling of triacylglycerols was inhibited. Thus, although labeled arachidonic acid was incorporated into phospholipids and triacylglycerols in brain subcellular membranes, these two types of acyltransferases exhibited different properties and responded differently to membrane perturbing agents.     

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-¹⁴C] arachidonate or [1-¹⁴C]oleate. During a 24-hr incubation, 25-or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-¹⁴C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.     

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine

The question of whether diplasmalogens [1,2-di(O-1′-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO₄ staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO₄ staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29±2 μg/10⁸ cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (¹H-NMR) spectroscopy, which showed the presence of O-1′-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104±5 μ/10⁸ cells, of which 50% was monoplasmalogen with the 1′-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.