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.     

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.     

Phospholipids inDrosophila heads: Effects of visual mutants and phototransduction manipulations

A procedure was developed to label phospholipids inDrosophila heads by feeding radioactive phosphate (³²P_i). High-performance thin-layer chromatography showed label incorporation into various phospholipids. After 24 h of feeding, major phospholipids labeled were phosphatidylethanolamine (PE), 47%; phosphatidylcholine (PC), 24%; and phosphatidylinositol (PI), 12%.Drosophila heads have virtually no sphingomyelin as compared with mammalian tissues. Notable label was in ethanolamine plasmalogen, lysophosphatidylethanolamine, lysophosphatidylcholine and lysophosphatidylinositol. Less than 1% of the total label was in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Other lipids labeled included phosphatidylserine, phosphatidic acid and some unidentified lipids. A time course (3–36 h) study revealed a gradual decrease in proportion of labeled PI, an increase in proportion of labeled PC and no obvious change in labeled PE. There were no significant differences in phospholipid labeling comparing theno receptorpotential (norpA) visual mutant and wild type under lightvs. dark conditions. However, overall³²P labeling was higher in the wild type fed in the light as compared to the dark and tonorpA either in light or dark. This suggests that functional vision facilitates incorporation of label. Differences in phospholipid labeling were observed between young and aged flies, particularly in lysophospholipids and poly-PI, implicating phospholipase A₂ function in recycling. Manipulations such as theouterrhabdomeresabsent andeyesabsent mutants and carotenoid deprivation failed to yield notable differences in phospholipid labeling pattern, suggesting that phospholipids important to vision may constitute only a minor portion of the total labeled pool in the head.     

Esterification of palmitic acid in swine aortic homogenates

The incorporation of [1-¹⁴C] palmitic acid into tissue lipids of the medial and intimal layers of swine aortic homogenates was investigated. The homogenates obtained were metabolically active as indicated by their ready incorporation of labeled palmitic acid into phospholipids, diglycerides and triglycerides in the presence of α-glycerophosphate in the incubation medium. Predominantly, labeling of phospholipids and especially of phosphatidylcholine was found when α-glycerophosphate or lysolecithin served as the fatty acid acceptor. Glycerol and monoolein did not serve as fatty acid acceptors. More than 98% of the radioactivity was recovered as the phosphatidylcholine fraction at the level of 0.64 μmoles/ml of lysolecithin in the incubation medium.     

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

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.     

Potentiating effect of 5,8,11-eicosatrienoic acid on human platelet aggregation

5,8,11-Eicosatrienoic acid (20∶3ω9), a fatty acid increased in the platelet phospholipids of man and animals fed saturated fats, was either added to human platelets simultaneously with the aggregating agents, or incorporated into the platelet phospholipids by preincubation. 20∶3ω9 markedly increased the response of platelets to all aggregating agents tested when added simultaneously with the agent, but solely to thrombin and ionophore, after incorporation into the platelet phospholipids. The potentiating effects of 20∶3ω9 on thrombin aggregation do not appear to be related to prostaglandin formation, but rather to the production of a monohydroxy derivative through the lipoxygenase pathway.     

Placental transport oftrans fatty acids in the rat

Placental transport of 9-trans [1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans [1-¹⁴C] octadecadienoic (linoelaidic) acids was demonstrated in rats. On the 18th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparison, 9-cis [1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis [1-¹⁴C] octadecadienoic (linoleic) acid also was injected into the maternal circulation of rats. All animals were sacrificed 1 hr following injection. Lipid composition and distribution of label were determined in maternal plasma, placental and fetal tissues. Differences in specific activities of plasma, placental and fetal total lipids indicated a decreasing concentration gradient for bothcis andtrans isomers of octadecenoic and octadecadienoic acids. Distribution of radioactivity in various lipid components was determined by thin layer chromatography. Irrespective of the label, the highest percentage of total radioactivity was carried by triglycerides (TG) in maternal plasma (∼60–80%), and was incorporated mainly in phospholipids (PL) of fetal tissues (∼50–60%). A nearly equal distribution of the label was found between PL and TG of placental lipids (∼40%). Radioactivity of fatty acid methyl esters (FAME) determined by radiogas liquid chromatography indicated that after injection of linoelaidate, radioactivity of maternal plasma, placental and fetal tissue FAME was associated only witht,t-18∶2. Following injection of elaidate, all the radioactivity in placental FAME was associated witht-18∶1; however, in fetal tissues, the label was distributed between 16∶0 andt-18∶1. These findings suggest that, in contrast to linoelaidic acid, rat fetal tissues can metabolize elaidic acid via β oxidation to form acetyl CoA and palmitic acid.     

Triacylglycerol structure of human colostrum and mature milk

Because triacylglycerol (TAG) structure influences the metabolic fate of its component fatty acids, we have examined human colostrum and mature milk TAG with particular attention to the location of the very long chain polyunsaturated fatty acid on the glycerol backbone. The analysis was based on the formation of various diacylglycerol species from human milk TAG upon chemical (Grignard degradation) or enzymatic degradation. The structure of the TAG was subsequently deduced from data obtained by gas chromatographic analysis of the fatty acid methyl esters in the diacylglycerol subfractions. The highly specific TAG structure observed was identical in mature milk and colostrum. The three major fatty acids (oleic, palmitic and linoleic acids) each showed a specific preference for a particular position within milk TAG: oleic acid for thesn-1 position, palmitic acid for thesn-2 position and linoleic acid for thesn-3 position. Linoleic and α-linolenic acids exhibited the same pattern of distribution and they were both found primarily in thesn-3 (50%) andsn-1 (30%) positions. Their longer chain analogs, arachidonic and docosahexaenoic acids, were located in thesn-2 andsn-3 positions. These results show that polyunsaturated fatty acids are distributed within the TAG molecule of human milk in a highly specific fashion, and that in the first month of lactation the maturation of the mammary gland does not affect the milk TAG structure.     

Fatty acid composition of subcellular particles from sheep platelets and topological distribution of phosphatidylethanolamine fatty acids in the plasma membrane

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18∶1; 32–41%), linoleic (18∶2, 10–17%), stearic (18∶0; 13–15%), palmitic (16∶0; 11–15%) and arachidonic (20∶4; 8–12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22∶0), 18∶0 and 16∶0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18∶1n−9. Our findings suggest that fatty acids are asymmetrically distributed between thecholineversus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzenesulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18∶2 and significantly less 18∶1 and 20∶5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturatedversus saturated PE acyl chains.     

Composition and positional distribution of fatty acids in phospholipids isolated from pork muscle tissues

The composition and positional distribution of fatty acids in phospholipids isolated from four locations of a hog carcass is presented. Variations in fatty acid composition of phospholipids were found depending upon the location in the carcass. The total unsaturated fatty acid content averaged 34.3 mole % for lecithin, 52.5 mole % for phosphatidylethanolamine, 40.3 mole % for phosphatidylserine and 41.3 mole % in sphingomyelin. The cephalins had a much higher percentage of polyunsaturated fatty acids than lecithin. The chief saturated fatty acid in lecithin and sphingomyelin was palmitic and in cephalins it was stearic. A snake venom enzyme preparation(Crotalus adamanteus) hydrolyzed primarily unsaturated fatty acids in phosphoglycerides and the higher the percentage of unsaturation within the fatty acid the higher percentage of hydrolysis occurred. The unsaturated fatty acids were found chiefly at the theβ-position and the saturated fatty acids at thea-position in the phosphoglycerides. Michigan State Agricultural Experiment Station Publication No. 3389. Supported by the U.S. Public Health Service Research Grant No. GM 08801-03.     

Cholesterol-cerebroside interaction: The role of α-hydroxy fatty acids

The esterification of cholesterol by the plasma phosphatidyl choline-cholesterol acyltransferase reaction was studied by two methods, radioisotopic and colorimetric, in the presence of cerebroside, ceramide, or methyl esters of lignoceric or α-hydroxy lignoceric acid. The radioisotopic method measures esterification of exogenous labeled cholesterol which must be taken up into the lipoprotein-bound pool prior to its utilization as a substrate. The colorimetric method measures esterification of endogenous lipoprotein-bound free cholesterol since the exogenous labeled cholesterol is negligible in concentration. Cerebroside and ceramide containing α-hydroxy fatty acids reduced the utilization of exogenous labeled cholesterol as substrate, but had no effect on lipoprotein-bound exogenous cholesterol esterification. Cerebroside and ceramide containing no α-hydroxy fatty acid had no effect on exogenous labeled cholesterol esterification. The methyl esters of lignoceric acid and α-hydroxy lignoceric acid had no effect on the esterification of exogenous cholesterol in plasma. There is a decrease in esterification of exogenous labeled cholesterol with increasing concentration of α-hydroxy fatty acid ceramide. Increasing the concentration of exogenous cholesterol tends to counteract the effect of the ceramide on cholesterol esterification. There was little effect on exogenous cholesterol esterification when the α-hydroxy fatty acid ceramide was exposed to plasma before adding the labeled cholesterol. The findings demonstrate an interaction between free cholesterol and cerebroside or ceramide containing α-hydroxy fatty acids, but the nature of the interaction is not elucidated.     

Regional distribution of tocopherols and fatty acids within soybean seeds

Seed coat, axis, and sections of cotyledons in three soybean cultivars were analyzed by high-performance liquid chromatography for tocopherols, and by gas-liquid chromatography for acyl lipids. Tocopherols were predominantly detected in axis, followed by cotyledons and seed coat. With a few exceptions, dominant components were γ- and δ-tocopherols, with much smaller amounts of α- and β-tocopherols. However, α-tocopherol was higher (P<0.05) for the Mikawajima cultivar than for Okuhara and Tsurunoko in all tissues. Triacylglycerols (TAG) were the major fraction of total lipids, representing 70% in axis and coat and 94% in cotyledons. A small difference (P<0.05) occurred in fatty acid composition of TAG when comparing seed coat to the axis. The fatty acid composition of phosphatidylinositol (PI) differed (P<0.05) from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in each tissue. Principally, the percentage of palmitic acid was higher, especially in axis and coat. In PE and PC, linoleic was greater, followed by palmitic, in all samples except for seed coat tissue in Mikawajima. The percentages of palmitic acid in both phospholipids were significant higher in the seed coat tissue from this cultivar than in cotyledon or axis of the other varieties. These results suggest that the differences in soybean cultivars could be appreciable, based on the distribution of tocopherols and fatty acids in each component part within soybean seeds.     

Incorporation of arachidonic acid and eicosapentaenoic acid into phospholipids by polymorphonuclear leukocytes in vitro

The present study demonstrated that the patterns of the incorporation of [1-¹⁴C] arachidonic acid and [1-¹⁴C] eicosapentaenoic acid into individual phospholipids by polymorphonuclear leukocytes were similar. However, human leukocytes exhibited higher activity than guinea pig periotoneal leukocytes in the formation of arachidonoyl- and eicosapentaenoyl-phosphatidic acid. Cells from both origins showed a decrease of label in phosphatidylcholine accompanied by an increase of label in phosphatidylethanolamine after a longer period (30–120 min) of incubation, suggesting that part of the arachidonoyl or eicosapentaenoyl moiety in phosphatidylethanolamine may be derived from that of phosphatidylcholine. The observed difference between human cells and elicited cells in the time-course of the incorporation of both fatty acids into phosphatidylcholine and phosphatidylethanolamine appears to be due to different contents of the diacyl and ether-linked class compositions of these phospholipids in cells from different origins. Both labeled fatty acids were incorporated more rapidly into the diacyl-linked class, but were retained to a greater extent in alkylacyl-phosphatidyl-choline and alkenylacyl-phosphatidylethanolamine. The data suggest that, in addition to alkylacyl-phosphatidylcholine and phosphatidylinositol, alkenylacyl-phosphatidylethanolamine may be an important endogenous source of arachidonic acid and eicosapentaenoic acid in stimulated human leukocytes.     

Human platelets release a paf-acether: Acetylhydrolase similar to that in plasma

Intact washed human platelets aggregated in response to paf-acether (paf) and did not metabolize [³H]paf at concentrations up to 10 nM. However, when platelets were lysed by exposure to pH 9.5, resulting in 37.5±2.5% (mean ±SD, n=3) lactic dehydrogenase (LDH) release, 20.5±5.7% of the radioactivity was detected as labeled lyso paf and 5.7±3.1% as labeled alkylacylglycerophosphocholine. When platelets were aggregated with 0.5 IU/mL thrombin or high concentrations of paf (100 nM), they released a part of their acetylhydrolase without releasing LDH. In supernatants obtained from aggregated platelets, 21±2% or 10±2% (n=3), respectively, of the total platelet acetylhydrolase activity was detectedvs. none in supernatants of resting cells. The release of acetylhydrolase was concentration-and time-dependent and paralleled the release of PF 4, a marker for α-granules. The acetylhydrolase affinity for paf (K_m) measured in sonicates of resting and thrombin-activated platelets was 8.3±1.5 μMvs. 10.6±1.5 μM, n=5, n.s. in a “Mann Whitney” test. The latter K_m was slightly but significantly different (P<0.05, n=5) from that of the thrombin-released acetylhydrolase (7.9 ±1.5 μM) and that of the latter was itself different from plasma acetylhydrolase (5.3±0.5,P<0.05, n=5). Addition of plasma (acid-treated to inactivate acetylhydrolase) decreased the K_m value of supernatant acetylhydrolase to 6.1±1.4 μM. All preparations of acetylhydrolase exhibited similar pH requirements and sensitvity to various inhibitors. Thus paf and thrombin cause release of acetylhydrolase from platelets in parallel with release of the α-granule marker PF4. This phenomenon might represent a protective mechanism against paf-mediated effects in thrombotic and cardiovascular diseases. This study has been presented in part as a preliminary report at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, NV, May 1988 (1).     

Production of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω-3) by a Δ5 and Δ12 desaturase-defective mutant of an arachidonic acid-producing fungus Mortierella alpina 1S-4

A Δ5 and Δ12 desaturase-defective mutant of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, produced 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) intracellularly when grown with linseed oil. Dihomo-γ-linolenic acid was the only C₂₀ polyunsaturated fatty acid (4.9 wt% of total mycelial fatty acids) other than 20:4ω3. AA and 5,8,11,14,17-cis-eicosapentaenoic acid were not detected. The mycelial lipids consisted of 82.2% (by mol) triacylglycerol (TG), 7.1% diacylglycerol, 8.9% phospholipids (PL), and 1.9% free fatty acids. The percentage of 20:4ω3 was higher in PL (30.1%) than in TG (11.6%), and highest in phosphatidylcholine (38.9%). Under the optimal conditions with a 5-L jar fermenter, 20:4ω3 production amounted to 97.4 mg/g dry mycelia with a mycelial yield of 23 g/L on the twelfth day (corresponding to 2.24 g/L medium and 37.1% of total mycelial fatty acids).     

Lung surfactant phospholipids in different animal species

A comparative study of adult mammalian lung surfactants was undertaken to determine which animal species might serve as appropriate models for surfactant alterations in human lung diseases. Phosphatidylcholine (PC) comprised 80–87% of the phospholipid and contained more than 65% palmitic acid in all species studied. Phosphatidylglycerol (PG) was found to vary significantly in fatty acid composition among the species. Rabbit, dog and rat surfactant PG contained 50–60% palmitic acid, while human and cat surfactant contained much lower levels of saturated fatty acids. Both the PC and PG of all species contained 2 positional isomers of fatty acids with 16 carbons and one double bond, but the relative amounts of the unusual isomer, 16∶1Δ7, and palmitoleic, acid, 16∶1Δ9, varied among the different animal species. Only cat and dog surfactant phospholipids contained 18∶1Δ5. Cat surfactant phospholipids also differed by the absence of 20∶4 and the presence of small amounts of several 20- and 22-carbon fatty acids. These results explain some discrepancies found in the literature concerning surfactant composition and delineate limiting factors in extrapolating results from animal studies for the evaluation of maturation and pathological alterations in human surfactant.     

Rat liver chromatin phospholipids

To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08–0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.     

Inhibitory effect of stearidonic acid (18∶4 n−3) on platelet aggregation and arachidonate oxygenation

The effect of stearidonic acid (18∶4n−3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18∶4n−3 appeared as a weak inhibitor of platelet aggregation. In addition, 18∶4n−3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18∶4n−3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20∶5n−3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20∶5n−3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18∶4n−3 may affect platelet aggregation by another mechanism.     

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.