Changes in free fatty acids and diglycerides in mouse brain at birth and during anoxia

Abstract: Within the first few hours of life in the mouse, marked changes were seen in brain endogenous free fatty acids (FFA). A 21% decrease in the total FFA pool occurred during the 1st h of life, and a constant value was maintained thereafter to 10 h. Polyunsaturated fatty acids displayed a different pattern of change. There was 27% less free ararhidonic acid at birth (0 h) than 1 h later. Similar values were obtained for docosahexaenoic acid at birth and at 10 h, although palmitoleic and oleic acids decreased markedly after 1 h. The polyunsaturated fatty acyl chains of diglycerides (DG) showed a statistically significant increase as a function of time after birth, despite an unchanged total DG pool size. The brains of pups subjected to 40 min of N₂-anoxia immediately after delivery exhibited a decrease in FFA, especially the monoenoic components, but 60 min of anoxia yielded higher FFA levels. Anoxia induced at 10 h increased FFA and arachidonic acid was higher than when anoxia was induced at 0 h. FFA accumulation was further stimulated by raising the environmental temperature during anoxia. When anoxia was induced, DG exhibited a net increase in palmitate, oleate, and palmitoleate at 0 and 10 h. No arachidonoyl-DG accumulated at 0 h, even after 60 min of anoxia, and stearate was unchanged at 0 and 10 h. The lipid changes observed in the brain during the first hours of life suggest that the enzymatic reactions that promote accumulation of free arachidonic and docosahexaenoic acids and arachidonoyl-DG in the mature brain are present at low levels at the time of delivery. The sluggish modifications found in our study may be related to the longer resistance of newborns to oxygen deficiency.     

Platelet-Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.     

α-Methyl-p-tyrosine inhibits the production of free arachidonic acid and diacylglycerols in brain after a single electroconvulsive shock

A single electroconvulsive shock (ECS) significantly enlarged the free fatty acid (FFA) pool of the mouse brain, arachidonic acid being the most actively released FFA (48% over controls). In animals pretreated with -methyl-p-tyrosine (-MT), the endogenous levels of FFA (25 sec after decapitation) were decreased and the effect of ECS was completely abolished. The most pronounced inhibition took place in free arachidonic acid (42% and 52% under controls in nonstimulated and ECS-stimulated mice, respectively). A similar tendency, although lower and less specific than the one taking place in FFA, was observed in mouse brain diacyl-glycerols (DG). In contrast to ECS, -MT treatment did not affect the marked release of FFA and DG taking place 3 min after decapitation. Taking into account the specific inhibitory action of -MT on tyrosine hydroxylase activity, the present findings provide experimental in vivo evidence about the relationship between biogenic amines and membrane lipid breakdown during electrical stimulation and suggest an involvement of FFA and DG in neurotransmission.     

Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

Brain Cortical Fatty Acids and Phospholipids During and Following Complete and Severe Incomplete Ischemia

Abstract: To explore the possibility that peroxtdative degradation of brain tissue lipid constituents is an important mechanism of irreversible ischemic damage, we measured cortical fatty acids and phospholipids during reversible brain ischemia in the rat. Neither complete nor severe incomplete ischemia (5 and 30 min) caused any measurable breakdown of total or individual fatty acids or phospholipids. Except for a small (and reversible) decrease of inositol plus serine phosphoglycerides in the early postischemic period following 30 min of incomplete ischemia, there were no significant losses of fatty acids or phospholipids during recirculation. Since peroxidation, induced in brain cortical tissue in vitro , characteristically involves degradation of polyenoic fatty acids (arachidonic and docosahexaenoic acids) and of ethanolamine phosphoglycerides, the present in vivo results fail to support the hypothesis that peroxidation of membrane lipids is of primary importance for ischemic brain cell damage. Both complete and severe incomplete ischemia caused a similar increase in the tissue content of free fatty acids (FFA). Thus the FFA pool increased by about 10 times during a 30-min ischemic period, to constitute 1 - 2% of the total fatty acid pool. Since there was a relatively larger increase in polyenoic FFA (especially in arachidonic acid) than in saturated FFA, the release of FFA may be the result of activation of a phospholipase A₂ unbalanced by reesterification. Increased levels of FFA persisted during the initial recirculation period, but a gradual normalization occurred and the ischemic changes were essentially reversed at 30 min after restoration of circulation. The pathophysiological implications of the changes in FFA are discussed with respect to mitochondrial dysfunction, formation of cellular edema and prostaglandin-mediated deterioration of postischemic circulation.     

Barbiturate Attenuation of Brain Free Fatty Acid Liberation During Global Ischemia

Abstract: To find a biochemical basis for the increased tolerance of the brain to anoxia during barbiturate anesthesia, we studied whole-brain free fatty acids (FFA) at various times after decapitation of awake and pentobarbital-anesthetized rats. Post-decapitation, the brains were kept at 37°C for 1 to 60 min before freezing in liquid N₂. Nonischemic brains were frozen in liquid N₂, using a rapid sampling technique. Whole-brain arachidonic, stearic, oleic, linoleic, and palmitic acids were quantitated by gas-liquid chromatography. In unanesthetized, nonischemic brain, total FFA was 1226 ± 121 nmol/g brain ( n = 12) and was unaffected by pentobarbital anesthesia (1126 ± 86 nmol/g brain, n = 11), except for a reduction in arachidonic acid. Total FFA in unanesthetized and pentobarbital-anesthetized rats transiently declined between 0 and 1 min of ischemia, and then rose linearly for up to 60 min, with consistently lower values in pentobarbital-treated rats, the greatest attenuation being that of arachidonic and stearic acid liberation. Brain FFA liberation during global ischemia is the first known biochemical variable directly correlated with the duration (i.e., severity) of global ischemia. The attenuation of brain FFA liberation and especially of arachidonic and stearic acids may be the biochemical basis of barbiturate attenuation of ischemic brain injury.     

Effect of the conditions of analysis and obtaining of the sample on the level of free fatty acids in the brain of the rat

Free fatty acids (FFA) have been determined in the rat brain by gas-liquid chromatography after isolation by thin-layer chromatography on silica gel. The brains were removed under three experimental conditions, 1) after freezing in situ with liquid nitrogen, 2) after immersion of the animal in liquid nitrogen, 3) after decapitation, the brain being frozen 3 minutes later. The total FFA level was found to be equal respectively to 20.1, 33.1 and 168 micrograms/g. In any case, the main fatty acids were palmitic, stearic and oleic acid but there were marked increases in arachidonic and docosahexaenoic acids following decapitation. A cause of error in the FFA determination originated in the use of commercial silica gel which contained significant amounts of fatty acids.     

Polyphosphoinositides as a Probable Source of Brain Free Fatty Acids Accumulated at the Onset of Ischemia

The quantitative relationship between phosphoinositides and free fatty acids (FFAs) in brain ischemia was studied by measuring contents of individual fatty acids in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DAG), and the FFA pool. Various periods of complete ischemia (1, 3, 10, and 30 min) were produced by decapitation. Ischemia of 1-3 min caused rapid decreases in PIP2 and PIP content together with preferential production of stearic and arachidonic acids in the DAG and FFA pools. The decrement in levels of these fatty acid residues in polyphosphoinositides was sufficient to account for their increment in levels in the enlarged DAG and FFA pools. After 10 min of ischemia, levels of PIP2, PIP, and DAG approached plateau values, but levels of all FFAs continued to increase. The increases in content of DAG and FFAs at later ischemic periods could not be accounted for by the decreases in content of PIP2 and PIP, PI and PA levels showed only transient and subtle changes. These results indicate that, at the onset of ischemia, phosphodiesteric cleavage of PIP2 and PIP and subsequent deacylation by lipases are primarily responsible for the preferential increase in levels of free stearic and arachidonic acids and that, later, hydrolysis of other phospholipids plays a major role in the continuous accumulation of FFAs.     

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.     

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Metabolic Turnover of Fatty Acids and Acylglycerols in Rat Sciatic Nerve

To explain the discrepancy between the low level and high metabolic activity of endoneurial free fatty acids (FFAs) and triacylglycerol (TG), levels of de novo synthesized FFA and acylglycerols were measured in rat sciatic endoneurium at various intervals after endoneurial microinjection of [14C]acetate. Soon after injection (less than 10 min), the [14C]acetate was metabolized to FFA and incorporated into diacylglycerol (DG), TG, sterols, ceramides, and various phospholipids. The proportions of 14C-labeled FFA, DG, TG, and ceramides to total 14C-labeled lipids decreased, whereas those of phospholipids and cerebrosides increased with time after injection. These findings suggest that rapid turnover of FFA and TG may contribute to their low level in sciatic endoneurium. The de novo synthesized fatty acids were largely incorporated into phosphatidylcholine (approximately 50% of total 14C-labeled phospholipids), probably via the cytidine nucleotide pathway using 1,2-DG as a metabolic intermediate. Hydrolysis of [14C]phosphatidylcholine revealed that fatty acids were labeled at both the C-1 (approximately 43%) and C-2 (approximately 57%) positions. On the other hand, a temporal association between decreased amounts of 14C-label in ceramides and increased amounts of 14C-label in sphingomyelin and galactocerebrosides supports the hypothesis that peripheral nerve galactocerebroside is derived, in vivo, from ceramide via acylation of sphingosine. This exclusive labeling of endoneurial lipids by endoneurial microinjection of labeled precursor provides a unique model for studying synthesis and metabolic turnover of membrane lipids in experimental neuropathies.     

Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.

Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzyme in vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.     

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [¹⁴C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A₂ activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PEpl ethanolamine plasmalogen - PI phosphatidylinositol - PS phosphatidylserine - poly-PI polyphosphoinsoitide - DG diacylglycerol - TG triacylglycerol - FFA free fatty acids - PUFA polyunsaturated fatty acids - MCA middle cerebral artery - CCAs common carotid arteries - HPTLC high performance thin layer chromatography - GLC gas-liquid chromatography - PLA₂ phospholipase A₂ Special issue dedicated to Dr. Leon S. Wolfe.     

Unsaturated Fatty Acids Esterified in 2-Acyl-1-Lysophosphatidylcholine Bound to Albumin Are More Efficiently Taken up by the Young Rat Brain than the Unesterified Form

The aim of the present study was to investigate whether unsaturated 2-acyl-lysophosphatidylcholine bound to plasma albumin is a relevant delivery form of unsaturated fatty acids to the developing brain. Twenty-day-old rats were perfused for 30 s with labeled palmitic, oleic, linoleic, and arachidonic acids in either their unesterified form or esterified in 2-acyl-lysophosphatidylcholine labeled on the choline and fatty acid moieties. Both forms were bound to albumin. Incorporation in brain lipid classes was followed within 1 h. The brain uptake of the unesterified fatty acids reached a plateau at 5-15 min and was maximal for arachidonic acid (0.45% of the perfused dose). The brain uptake of palmitoyl-lysophosphatidylcholine was similar to that of palmitic acid, whereas that of other lysophosphatidylcholines increased with the degree of unsaturation (rate and maximal uptake) and was six- to 10-fold higher than that of the corresponding unesterified fatty acid. 2-Acyl-lysophosphatidylcholines were taken up without prior hydrolysis and reacylated into doubly labeled phosphatidylcholine, which was the most labeled lipid class, whereas lipid distribution of the unesterified fatty acid was more diversified. Partial hydrolysis of 2-acyl-lysophosphatidylcholine occurred in the brain tissue, and redistribution of the fatty acyl moiety into other phospholipid classes was also observed and was the highest for arachidonic acid. In this case, the percentage of esterification of this fatty acid in phosphatidylinositol (expressed as a percentage of the total lipid fraction) was relatively lower than that observed when the unesterified form was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lesions to the Corticostriatal Pathways Ameliorate Hypoglycemia-Induced Arachidonic Acid Release

The concentrations of free fatty acids (FFAs) in the neostriatum of control rats and rats subjected to unilateral cortical ablation were measured during and following severe insulin-induced hypoglycemia. The total FFA concentration in the caudate nucleus contralateral to the lesion increased to approximately 1.5 and 3 times the control level after 5 and 30 min of isoelectricity, respectively, and was similar to the control value following 1 h of recovery. After 5 min of isoelectricity, the total FFA pool was significantly smaller in the decorticated striatum. No difference between hemispheres was noted after 30 min of isoelectricity. After 5 min of isoelectricity the levels of stearic and arachidonic acid were selectively increased whereas palmitic acid and oleic acid remained at control levels. In the decorticated striatum of lesioned animals the arachidonic acid concentration was significantly lower, whereas the level of stearic acid was not significantly different from the control value. After 30 min of isoelectricity the levels of all four FFA species were increased. Apart from a significantly lower level of oleic acid on the decorticated side, there were no interhemispheric differences in the FFA levels. Since the early interhemispheric differences in the FFA levels. Since the early interhemispheric hemispheric differences in the levels of arachidonic and stearic acids coincide with a selective decrease in the levels of glutamate and a decreased energy utilization on the decorticated side, the results suggest that glutamate release during hypoglycemia induces an early receptor-mediated degradation of phospholipids, presumably via the phosphatidylinositol cycle.     

Regional Studies of Changes in Brain Fatty Acids Following Experimental Ischaemia and Reperfusion in the Gerbil

Regional studies of brain phospholipid metabolism were carried out during a period of ischaemia induced in the gerbil by bilateral carotid occlusion for 60 min. The associated changes in free fatty acids (FFAs) during this period and following recirculation for up to 180 min were noted. Following ischaemia there was a generalised rise in the levels of all FFAs with no selective release of either the unsaturated (arachidonic and docosahexaenoic) or saturated (palmitic and stearic) fatty acids. There were no observed differences between the brain regions studied, which is in contrast to previously reported observations for prostaglandins. There was also no indication of any specific phospholipid fraction being involved in FFA release. This would indicate that the release of FFAs from phospholipids is a nonspecific event, probably due to the action of hydrolytic lipases. Restoration of the circulation resulted in a short, sharp increase (within 5 min) in all FFAs, but in contrast to the observations during ischaemia alone there was a relatively larger rise in the unsaturated FFAs as compared to the saturated FFAs. Following this increase there was a gradual general decline in all FFA levels until 180 min of reperfusion. Since there was no preferential depletion of unsaturated FFAs during reperfusion, when free radical attack is considered to be at its maximum, it is our opinion that free radical peroxidation is unlikely to explain the pathology described in our model.     

Diacylglycerol Kinase Is Stimulated by Arachidonic Acid in Neural Membranes

Abstract: The effect of arachidonic acid (AA) on the activity of diacylglycerol (DG) kinase in neural membranes was investigated. When rat brain cortical membranes were incubated with 0.5 m M dipalmitin and [γ-³²P]ATP, formation of phosphatidic acid (PA) was observed. It was linear up to 5 min, and the initial rate was ∼1.0 nmol/min/mg of protein. The DG kinase activity was stimulated twofold by 0.25 m M AA. The stimulation was apparent at the earliest time point measured (1 min) and with the lowest concentration of AA tested (62.5 µ M ). The stimulation was proportional to the concentration of AA up to 250 µ M . AA was the most potent stimulator of DG kinase, and linolenic acid showed ∼40% stimulation. Oleic acid showed no effect, whereas linoleic and the saturated fatty acids tested were inhibitory. AA stimulation of DG kinase was observed only with membranes of cerebrum, cerebellum, and myelin and not with brain cytosol or liver membranes. AA also stimulated the formation of PA in the absence of added dipalmitin (endogenous activity) with membranes prepared from whole brain. DG kinase of neural membranes was extracted with 2 M NaCl, which on dialysis yielded a precipitate. Both the precipitate and the supernatant showed DG kinase activity, but only the enzyme in the precipitate was stimulated by AA at concentrations as low as 25 µ M . It is suggested that AA, through its effect on DG kinase, regulates the level of DG in neural membranes, which in turn regulates protein kinase C activity.     

The effect of arginine vasopressin on the excitability and fatty acid content of the brain structures in rats with a neurosis

Experiments on rats with chronic bipolar electrodes implanted into the frontal cortex (FC), dorsal hippocampus (DH) and midbrain reticular formation (RF) established that the neurotic state (model "conflict of afferent excitation") was characterised by the increase in structure excitability: FC--15.4% (P less than 0.01), DH--12.4% (P = 0.05) and RF--17.5% (P less than 0.001). The presence of free fatty acids (FFA) revealed by chromatograph Schimadzu in brain cortex (BC), hippocampus (H) and midbrain (MB) in acute experiments evidenced the increase in the level of linolinic acid in all matters within the limits of 64-162% (P less than 0.05) and also different changes in arachidonic acid in BC and subcortical structures. The level of arachidonic acid increased by 120% (P less than 0.01) in BC but it decreased in H and MB within the limits of 34-56%. AVP (1 micrograms/kg) decreased excitability of FC by 6% (P less than 0.001), of H--by 8% (P less than 0.01) and RF--by 6%. In this case FFA, especially arachidonic acid, was increased in H and MB (by 2.5-6 times). The quantity of palmitic, stearic and oleic acids increased.     

Biphasic Liberation of Arachidonic and Stearic Acids During Cerebral Ischemia

The mode of free fatty acid (FFA) liberation from the mouse brain during ischemia was investigated at various times after decapitation and under nizofenone treatment. Normal nonischemic brain FFAs consist mainly of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) with smaller amounts of arachidonic acid (20:4), docosahexaenoic acid (22:6), and others. Postdecapitative ischemia induced a rapid, biphasic release of 20:4 after a short lag of less than 30 s. The first phase showed a rapid 6.4-fold increase within 1 min of decapitation, followed by the second phase involving a slow release at less than one-fifth the rate of the first phase and lasting for at least 10 min. A similar, but not so marked, biphasic liberation was observed with 18:0. However, all of the other fatty acids (16:0, 18:1, 22:6, and others) were released only in a single phase at a slow rate. The time course for the rapid and specific liberation of 20:4 coincided with the time course for the decrease in brain ATP concentration during ischemia. Pretreatment of the animals with nizofenone resulted in a marked suppression of both FFA liberation and ATP depletion during ischemia. This suppression was particularly noteworthy with 20:4 and 18:0. The present study indicates that there is a specific and rapid liberation of 20:4 and 18:0 in a very early stage of ischemia and that this liberation seems to depend on availability of ATP in the brain. The physiological role of this transient 20:4 liberation during ischemia is discussed.