Alterations in the Fatty Acid Composition of Rat Brain Cells (Neurons, Astrocytes, and Oligodendrocytes) and of Subcellular Fractions (Myelin and Synaptosomes) Induced by a Diet Devoid of n-3 Fatty Acids

Abstract: Rats were fed through four generations with a semisynthetic diet containing 1.0% sunflower oil (6.7 mg/ g n-6 fatty acids, 0.04 mg/g n-3 fatty acids). Ten days before mating, half of the animals received a diet in which sunflower was replaced by soya oil (6.6 mg/g n-6 fatty acids, 0.8 mg/g n-3 fatty acids) and analyses were performed on their pups. Fatty acid analysis in isolated cellular and subcellular material from sunflower-fed animals showed that the total amount of unsaturated fatty acids was not reduced in any cellular or subcellular fraction (except in 60-day-old rat neurons). All material from animals fed with sunflower oil showed an important reduction in the docosahexaenoic acid content, compensated (except in 60-day-old rat neurons) by an increase in the n-6 fatty acids (mainly C22:5 n-6). When comparing 60-day-old animals fed with soya oil or sunflower oil, the n-3/n-6 fatty acid ratio was reduced 16-fold in oligodendrocytes, 12-fold in myelin, twofold in neurons, sixfold in synaptosomes, and threefold in astrocytes. No trienes were detected. Saturated and monounsaturated fatty acids were hardly affected. This study provides data on the fatty acid composition of isolated brain cells.     

Effect of exogenous gangliosides on the lipid composition of chick neurons in culture

When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior. The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture. The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids. The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides. The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels. The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation. Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:6ω-3) and Arachidonic Acid (20:4ω-6)

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.     

Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells

The cellular composition and concentration of fatty acids are crucial for proliferation and survival. We recently showed stimulation of protein phosphatase type-2C (PP2C) by unsaturated fatty acids. Here, we describe that treatment of cultured chick neurons with 100 microM oleic acid for 24h increased the percentage of damaged neurons to 61+/-9% compared with 25+/-4% in controls. Oleic acid-induced cell death showed features of apoptosis such as chromatin condensation, shrinkage of the nucleus, DNA fragmentation and caspase-3 activation. Extensive studies with a variety of fatty acids revealed a striking correlation between activation of PP2C and induction of apoptosis. Lipophilicity, oxidizability, and an acidic group were required for both effects. In addition, activation of PP2C and induction of apoptosis could discriminate between cis- and trans-conformation of the fatty acids. The results are in favor of PP2C playing an important, yet unidentified role in apoptosis.     

Gangliosides of human, bovine, and rabbit plasma

Gangliosides were isolated from human, bovine, and rabbit plasma and were quantified by gas-liquid chromatography. Purification was achieved by sequential use of partitioning in solvents, DEAE-Sephadex chromatography, base treatment, and silicic acid chromatography. Human and bovine plasma yielded slightly more than 1 micro mole of lipid-bound sialic acid/100 ml; for rabbit plasma the value was 0.28 micro mole/100 ml. The total bovine plasma ganglioside fraction contained equal amounts of N-acetylneuraminic and N-glycolylneuraminic acids, rabbit plasma gangliosides had about 1% of the latter, and the human plasma sample contained only the former. Thin-layer chromatography revealed important differences among the plasmas from the three species, but all possessed hematosides and hexosamine-containing gangliosides. The approximate ratios of these two categories, based on sialic acid content, were (hematosides: hexosamine-type): human, 2:1; rabbit, 3:2; and bovine, 2:3. The fatty acid compositions of both categories were characteristic of extraneural gangliosides and included six major acids: palmitic, stearic, behenic, tricosanoic, lignoceric, and nervonic. The major long-chain base in each sample was sphingosine, while only a trace of the C(20) isomer was detected.     

Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes

DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.     

Sulfatide with short fatty acid dominates in astrocytes and neurons

Glycosphingolipids are located in cell membranes and the brain is especially enriched. We speculated that the subcellular location of glycosphingolipids depends on their fatty acid chain length because their sugar residues are constant, whereas fatty acid chain length can vary within the same molecule. To test this hypothesis we analysed the glycosphingolipid sulfatide, which is highly abundant in myelin and has mostly long fatty acids. We used a negative ion electrospray tandem mass spectrometry precursor ion scan to analyse the molecular species of sulfatide in cultured astrocytes and a mouse model of the human disease metachromatic leukodystrophy. In these arylsulfatase A (ASA)-deficient mice sulfatide accumulates intracellularly in neurons and astrocytes. Immunocytochemistry was also performed on cultured astrocytes and analysed using confocal laser scanning microscopy. Analyses of the molecular species showed that cultured astrocytes contained sulfatide with a predominance of stearic acid (C18), which was located in large intracellular vesicles throughout the cell body and along the processes. The same was seen in ASA-deficient mice, which accumulated a higher proportion (15 mol% compared with 8 mol% in control mice) of sulfatide with stearic acid. We conclude that the major fatty acid composition of sulfatide differs between white and grey matter, with neurons and astrocytes containing mostly short-chain fatty acids with an emphasis on stearic acid. Based on our results, we speculate that the fatty acid chain length of sulfatide might determine its intracellular (short chain) or extracellular (long chain) location and thereby its functions.     

Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.     

Comparative utilization of n-3 polyunsaturated fatty acids by cultured human Y-79 retinoblastoma cells

The Y-79 retinoblastoma cell, a cultured human line derived from the retina, was utilized as a model for investigating the metabolism of n-3 polyunsaturated fatty acids in neural tissue. When cultures were incubated with 5 microM linolenic (18:3), eicosapentaenoic (20:5) or docosahexaenoic (22:6) acids, a low concentration probably representative of physiologic levels, the amount incorporated was 20:5 congruent to 18.3 greater than 22:6. Regardless of which fatty acid was provided, 65-75% of the total uptake accumulated in phosphatidylethanolamine and ethanolamine plasmalogen, suggesting that these phospholipids play an important role in n-3 polyunsaturated fatty acid metabolism. A small amount of 22:6 was converted to 20:5, which was recovered in phosphatidylinositol and phosphatidylserine. Therefore, one metabolic function of 22:6 may be to serve as an intracellular storage pool for the formation of 20:5 through retroconversion. When any of the n-3 polyunsaturates was available, the main fatty acid that accumulated in the cell phospholipids was 22:6. The extent to which 22:6 accumulated, however, depended on the particular n-3 polyunsaturated fatty acid that was available. This suggests that the 22:6 content of a neural cell, and any cellular function dependent on 22:6 content, may be regulated by changes in the type of n-3 polyunsaturate available to the nervous system.     

Synthesis of hydroxy fatty acids from 4, 7, 10, 13, 16, 19-[1-14C] docosahexaenoic acid by human platelets

Human platelets incubated in the presence of 54 microM [1-14C]22:6 produced hydroxydocosahexaenoic acid (HDHE) at about half the rate with which 12-hydroxy-5,8,10,14-eicosatetraenoic acid is produced from [1-14C]arachidonic acid. More than 90% of the radioactivity in HDHE was distributed among two major isomers, 14-HDHE and 11-HDHE. The production of HDHEs was unaffected by indomethacin but completely inhibited by 5,8,11,14-heneicosatetraynoic acid, which suggests that the hydroxy fatty acids are produced by lipoxygenase. The proportions of HDHE isomers varied with the concentration of 22:6. The ratio 14-HDHE/11-HDHE was higher at 6.8 microM 22:6 than when platelets were incubated with 54 microM 22:6. It is suggested that the amounts of these isomers produced will depend both on the availability of 22:6 as well as by competition of this acid with other acids for lipoxygenase.     

Effect of alcohol on the metabolic conversion of 14C erucic acid in the liver of rats receiving rapeseed or peanut oil]

The effects of addition of ethanol to diets containing rapeseed or ground nut oil on the metabolic conversions of 14 14C erucic and 9-10 3H oleic acid were studied in the rat liver. 1. Whatever the diet more 14C than 3H radioactivity was recovered in liver lipids 2 and 19 hours after injection of labelled fatty acids. Ethanol has little effect on this incorporation. 2. Only small amounts of 3H oleic acid were converted. 3. In all cases, the metabolic conversion of erucic acid was identical: the main part of 14C was not recovered as erucic acid but was present in other monounsaturated fatty acids n-9: oleic acid (18 : 1), which was the most labelled acid, 16 : 1, 20 : 1 and nervonic acid (24 : 1). 4. The amount of erucic acid converted to shorter chain fatty acids was unchanged by addition of ethanol but the alcohol increased the proportion of 14C radioactivity recovered as nervonic acid. This latter effect was opposite to the effect of rapeseed oil diet, which consisted in a decrease in the conversion of erucic to nervonic acid. 5. A high amount of 14C radioactivity was recovered in the F.F.A. fraction of the liver as an unknown compound (13 and 80% of 14C radioactivity respectively after 2 and 19 h.) Its identification is presently under investigation.     

Ultrastructural and biochemical studies of the swelling of developing chick telencephalic slices

Summary An ultrastructural and biochemical study of the importance and localization of tissue swelling was performed on telencephalic slices of 1- and 30-day-old chicks incubated in an oxygenated or a non-oxygenated physiological medium. The swelling of slices is greater for 30-day-old chick material than for that from 1-day-old chicks. It also reaches higher values in the non-oxygenated than in the oxygenated medium. When the 30-day-old chick telencephalic slices are incubated in an oxygenated medium, swelling mainly affects astrocytes, and especially the astrocytic endfeet. When they are incubated in a non-oxygenated medium, the astrocytes and astrocytic endfeet are very swollen and in addition the swelling also affects the neurons and their organelles. Extracellular space is increased. When 1-day-old chick telencephalic slices are incubated in a non-oxygenated medium, the tissue structures are well preserved. Swelling predominantly affects astrocytes and astrocytic endfeet. Neurons are not affected and the extracellular space is reduced. However, when they are incubated in an oxygenated medium, tissue structures are greatly affected showing a high degree of disorganization. Extracellular space is greatly increased. This study thus indicates that the best incubation conditions are an oxygenated medium for 30-day-old chick telencephalic slices which are characterized by an aerobic metabolism, and a non-oxygenated medium for 1-day-old chick telencephalic slices which have a predominantly anaerobic metabolism.     

Glutamine Transaminase K and ω-Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching

Abstract: Glutamine transaminase K and ω-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of gluta-mine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (< 15-fold). Cerebral ω-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and ω-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of ω-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate β-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.     

Fatty acid composition and thermal behavior of natural sphingomyelins

We found significant differences in the fatty acid composition of several bovine brain, egg yolk and sheep erythrocyte sphingomyelins. These differences in fatty acid composition influence the thermal behavior of hydrated sphingomyelin as recorded by differentail scanning calorimetry. Significant differences were also found in the temperature and complexity of the order-disorder phase transitions of bovine brain sphingomyelin obtained from different sources which, in general, correlate with the relative content of the saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0) acids) and the long unsaturated nervonic acid (C24:1).     

Adrenic acid content in rat adrenal mitochondrial phosphatidylethanolamine and its relation to ACTH-mediated stimulation of cholesterol side chain cleavage reaction

We have isolated various phospholipids from adrenal mitochondria of adrenocorticotropic hormone (ACTH)-treated (stimulated) and cycloheximide/ACTH-treated (unstimulated) rats. When the effects of these phospholipids were examined on the formation of pregnenolone from endogenous cholesterol by adrenal mitochondria of unstimulated rats, phosphatidylethanolamine and phosphatidylserine from stimulated mitochondria were effective in enhancing the cleavage reaction in unstimulated mitochondria, whereas these phospholipids from unstimulated mitochondria were all ineffective. Cardiolipins from both stimulated and unstimulated mitochondria were effective. When the compositional changes in fatty acid moiety of phospholipids were examined, a significant increase in C22:4 (adrenic) acid was observed only for phosphatidylethanolamine under the influence of ACTH. A linear relationship between the contents of C22:4 acid in various phospholipids and respective steroidogenic activities was obtained (r = 0.880), suggesting an important role of this fatty acid moiety. The separation of active phosphatidylethanolamine by high performance liquid chromatography revealed that a fraction containing 25% C22:4 acid was most effective in the activation. Based on these results, it is most likely that 1-stearoyl-2-adrenoyl phosphatidylethanolamine is an active species. C22:4 acid was liberated together with C20:4 acid from adrenal triglycerides by the action of ACTH but the liberation was insensitive to cycloheximide inhibition. Finally, cardiolipin which enhances the transfer of cholesterol to cytochrome P-450scc may not be a physiological mediator of ACTH action.