Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been used to study accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl3-CH3OH-H2O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacyl-sn-glycero-3-phosphocholine (PCDIACYL) at -0.84 ppm). These signals were identified as originating from 1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (NAPEDIACYL) and 1-(1'-alkenyl)-2-acyl-sn -glycero-3-phospho-(N-acyl)-ethanolamine (NAPEPLAS), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N-acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with K-EDTA.The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPEDIACYL and NAPEPLAS) in rat brains by the use of 31P NMR spectroscopy.     

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.     

Biosynthetic pathways of the endocannabinoid anandamide

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.     

Substantial species differences in relation to formation and degradation of N-acyl-ethanolamine phospholipids in heart tissue: an enzyme activity study

The formation of N-acyl-ethanolamines (NAEs), including the cannabinoid receptor ligand anandamide, and their precursors N-acyl-ethanolamine phospholipids (NAPEs) are catalyzed by NAPE-hydrolyzing phospholipase D (NAPE-PLD) and N-acyl-transferase, respectively. NAPE and NAE are suggested to have beneficial effects on the heart, but in the literature there are indications of species differences in the activity of these enzymes. We have examined heart microsomes from rats, mice, guinea pigs, rabbits, frogs, cows, dogs, cats, mini pigs and human beings for activities of these two enzymes. N-Acyl-transferase activity was very high in dogs and cats (>13 pmol/min/mg protein) whereas it was very low to barely detectable in the other species (<3 pmol/min/mg protein). NAPE-PLD activity was very high in rats and guinea pigs (>45 pmol/min/mg protein) whereas it was 9 pmol/min/mg protein in frogs and below that in the other species. The ratio of activity between the two enzymes varied from 0.002 to 15 in the investigated species. The activity of the two enzymes in rat hearts as opposed to rat brain did not change during development. These results indicate that there may be substantial species differences in the generation of anandamide and other NAEs as well as NAPEs in heart tissues.     

Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA₁ and PLA₂. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA₁ hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA₁ only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues.     

Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids

N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. Like other lipid transmitters, NAEs are thought to be biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, we describe the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene [NAPE-PLD(-/-) mice]. Brain tissue from NAPE-PLD(-/-) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(-/-) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains (>or=C20). Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole.     

Formation of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ε in an ex vivo murine model of brain ischemia

N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A₂ε (cPLA₂ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA₂ε using cPLA₂ε-deficient (Pla2g4e^?/?) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca²⁺-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e^?/? mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e^?/? mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA₂ε is responsible for Ca²⁺-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.     

Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines

Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical “transacylation-phosphodiesterase” pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca²⁺-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A₂ enabled the further molecular biological approaches toward this enzyme. In addition, Ca²⁺-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1–5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4 and GDE7) as well as α/β-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners

N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to lecithin-retinol acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylcholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.     

Generation of N-Acylphosphatidylethanolamine by Members of the Phospholipase A/Acyltransferase (PLA/AT) Family

Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.     

N-acylation of phosphatidylethanolamine and its biological functions in mammals

N-acylphosphatidylethanolamine (NAPE) and N-acylplasmenylethanolamine (pNAPE) are widely found phospholipids, and they are precursors for N-acylethanolamines, a group of compounds that has a variety of biological effects and encompasses the endocannabinoid anandamide. NAPE and pNAPE are synthesized by the transfer of an acyl chain from a donor phospholipid, to the amine in phosphatidylethanolamine or plasmenylethanolamine. NAPE has been reported to stabilize model membranes during brain ischemia, and to modulate food intake in rodents, thus having bioactive effects besides its precursor role. This paper reviews the metabolism, occurrence and assay of NAPE and pNAPE, and discusses the putative biological functions in mammals of these phospholipids. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Catalytic Properties of a Newly Discovered Acyltransferase That Synthesizes N-Acylphosphatidylethanolamine in Cottonseed (Gossypium hirsutum L.) Microsomes

We recently demonstrated that cotyledons of cotton (Gossypium hirsutum L.) seedlings synthesize N-acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), during postgerminative growth (K.D. Chapman and T.S. Moore [1993] Arch Biochem Biophys 301: 21-33). Here, we report the discovery of an acyltransferase enzyme, fatty acid: diacylphosphatidylethanolamine N-acyltransferase (designated NAPE synthase), that synthesizes NAPE from PE and free fatty acids (FFA) in cottonseed microsomes. [14C]NAPE was synthesized from [14C]palmitic acid and endogenous PE in a time-, pH-, temperature-, and protein concentration-dependent manner. [14C]Palmitic acid was incorporated exclusively into the N-acyl position of NAPE. [14C]palmitoyl coenzyme A (CoA) and [14C]-dipalmitoyl phosphatidylcholine (PC) were poor acyl donors for the synthesis of NAPE (i.e. 200- and 3000-fold lower incorporation efficiency than palmitic acid, respectively). Synthesis of NAPE from palmitoyl-CoA and dipalmitoyl-PC was observed only after the release of FFA in microsomes. We observed a temperature optimum of 45[deg]C and a pH optimum of 8.0 for the synthesis of [14C]NAPE from [14C]palmitic acid (or from [14C]PE). NAPE synthase activity showed no apparent divalent cation requirement. Notably, activity was stimulated by HPO42-, HCO3-, SO42-, and NADPH, whereas activity was inhibited by Ca2+, Mn2+, Cd2+, ATP, ADP, flavin adenine disnucleotide, and flavin mononucleotide. Other nucleotide triphosphates (GTP and CTP) and pyridine dinucleotides (NAD, NADH, and NADP) did not appreciably affect NAPE synthase activity. Initial velocity measurements of NAPE synthase activity at increasing concentrations of palmitic acid showed non-Michaelis-Menten, biphasic kinetics. A high-affinity site (S0.5 = 7.2 [mu]M, Vmax = 18.8 nmol h-1 mg-1 of protein) and a low-affinity site (S0.5 = 32.0 [mu]M, Vmax = 44.9 nmol h-1 mg-1 of protein) were identified. Both sites exhibited positive cooperativity. Adding myristic, stearic, or oleic acids at equimolar amounts reduced the incorporation of [14C]palmitic acid into NAPE at low concentrations (10 [mu]M, high-affinity site) but not at high concentrations (50 [mu]M, low-affinity site), indicating that the two putative sites can be distinguished by their fatty acid preferences.     

Accumulation of the anandamide precursor and other N-acylethanolamine phospholipids in infant rat models of in vivo necrotic and apoptotic neuronal death

It has been demonstrated that the endogenous cannabinoid receptor ligand, anandamide, and other N-acylethanolamines (NAEs), accumulate during neuronal injury in vitro, a process that may be linked to the neuroprotective effects of NAEs. The crucial step for generation of NAEs is the synthesis of the corresponding precursors, N-acylethanolamine phospholipids (NAPEs). However, it is unknown whether this key event for NAE formation is regulated differently in the context of insults causing necrotic or apoptotic neuronal death. To address this question, we monitored a range of cortical NAPE species in three infant rat models of in vivo neurodegeneration: (i) necrosis caused by intrastriatal injection of NMDA (25 nmol); (ii) apoptosis induced by systemic administration of the NMDA-receptor antagonist (+)MK-801 (3 x 0.5 mg/kg, i.p.); and (iii) apoptosis following focal necrosis triggered by concussive head trauma. A marked increase of all NAPE species was observed in both hemispheres 4 and 24 h after NMDA-induced injury, with a relatively larger increase in N-stearoyl-containing NAPE species. Thus, the percentage of the anandamide precursor fell from 1.1 to 0.5 mol %. In contrast, administration of (+)MK-801 did not alter cortical NAPE levels. Concussion head trauma resulted in a similar but less pronounced upregulation of NAPE levels at both 4 and 24 h as compared to NMDA injections. Increased levels of NAPE 24 h post-trauma possibly reflect that necrosis is still ongoing at this time point. Consequently, our data suggest that excitotoxic necrotic mechanisms of neurodegeneration, as opposed to apoptotic neurodegeneration, have a profound effect on in vivo NAE precursor homeostasis.     

Basis for Phospholipid Incorporation into Peripheral Nerve Myelin

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P₀ (Golgi) and myelin basic proteins (more direct).     

Changes of phospholipid composition and superoxide dismutase activity during global brain ischemia and reperfusion in rats

Alterations in phospholipid content and Cu/Zn superoxide dismutase (SOD) activity were examined in rat brain after 15 min of global ischemia (four-vessel occlusion) followed by 2-, 24- or 48-h reperfusion. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the main brain phospholipids, were markedly decreased in ischemic rats and remained decreased during the whole reperfusion period. Concentrations of phosphatidylinositol (PI) and sphingomyelin (SM) were also significantly reduced during ischemia but recovered during reperfusion period. In contrast, phosphatidylserine (PS) and lysophospholipids (LysoPL) were unchanged during ischemia but were elevated after 24 h of reperfusion. Significant reductions in blood plasma phospholipids were also demonstrated. 24-48 h of reperfusion markedly decreased PE, PC and PS contents, while the concentrations were almost unchanged by ischemia alone. Brain SOD activity decreased significantly during ischemia and was recovered to control value already after 2 h of reperfusion. These results suggest that ischemia/reperfusion is accompanied by a significant and selective degradation of brain phospholipids that may be attributable to oxidative stress and activation of phospholipases.     

MODIFICATIONS IN SOLUBLE PROTEIN KINASE AND CYCLIC-AMP BINDING CAPACITY OF DEVELOPING RAT BRAIN

Histone and casein phosphoprotein-kinase activities were determined in rat brain soluble fraction at various stages of development. Cyclic AMP -independent or basal histone kinase activity increased, whereas cyclic AMP -dependent activity decreased in whole soluble fraction with the age. On the contrary, whole soluble cyclic AMP -dependent and -independent casein kinases activities did not show any difference during development. The percentage of activation by cyclic AMP of histone kinase activity and [³H] cyclic- AMP binding activity in the soluble fraction decreased markedly during development. By DEAE-cellulose chromatography the histone kinase was separated mainly into 4 peaks; the fourth peak was strongly stimulated by cyclic AMP . Stimulation by cyclic AMP was higher in the 4-day-old rat brains than in the 9- and 30-day-old. In the 9-day-old rats the ratio of cyclic AMP -dependent histone kinase in respect to the cyclic AMP -independent enzyme was higher than in 4- and 30-day-old rats. Casein kinase activities in the brains of 9- and 30-day-old rats were separated by DEAE-cellulose chromatography into three peaks of which the third one was stimulated by cyclic AMP . Little, if any, difference was observed for casein kinase during the development. These results suggest that brain histone and casein kinase are different enzymes:     

N-Acylation of ethanolamine phospholipids by acyl transfer does not involve hydrolysis

N-Acylethanolamine phospholipids occur in infarcted but not in normal canine myocardium. Their synthesis is catalyzed by a membrane-bound, Ca2+-requiring N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of various phospholipids including phosphatidylethanolamine to the amino group of ethanolamine phospholipids. When dog heart mitochondria are incubated in media containing Ca2+ and H2(18)O, the resulting N-acylethanolamine phospholipids do not accumulate 18O in either the amide or 1-O-acyl groups. The results indicate that acyl transfer occurs without hydrolysis, most likely through an acyl-enzyme complex which may be covalently linked.     

CARBOHYDRATE AND ENERGY METABOLISM IN PERINATAL RAT BRAIN: RELATION TO SURVIVAL IN ANOXIA

The ability of rats of different ages to survive exposure to anoxia was correlated with rates of high energy phosphate consumption (metabolic rates) of the fore-brain. Fetal rats at term, delivered by hysterotomy following maternal decapitation, survived in nitrogen at 37°C twice as long as 1-day-old neo-nates, 5 times longer than 7-day-old rats, and 45 times longer than adults. During ischemia induced by decapitation, the cerebral concentrations of the labile energy reserves (ATP, ADP, P-creatine, glucose and glycogen) and of lactate were determined in fetuses, 1- and 7-day post-natal animals. From the changes, the cerebral energy use rates were calculated to be 1·57 mmol/kg/min in fetuses, 1·33 mmol/kg/min in 1-day-olds and 2·58 mmol/kg/min in 7-day-olds. Maximal rates of lactate accumulation during ischemia, as a measure of glycolytic capacity, were comparable in fetuses and neonates, but were about twice as great in 7-day-old rats. It is concluded that in post-natal animals survival in anoxia and cerebral energy consumption are inversely, and nearly quantitatively, related. However, the reduced cerebral energy requirement cannot entirely account for the greater anoxic resistance of fetuses.     

Determination of the phospholipid precursor of anandamide and other N-acylethanolamine phospholipids before and after sodium azide-induced toxicity in cultured neocortical neurons

Phospholipase D-mediated hydrolysis of N-acylethanolamine phospholipids (NAPEs) releases anandamide and other N-acylethanolamines, resulting in different actions at cellular targets in the CNS. Recently, we have demonstrated that these N-acyl lipids accumulate in cultured neocortical neurons subjected to sodium azide-induced cell injury. We here extend the information on the NAPE response, reporting on the composition of N-acylspecies of NAPE, employing a new methodological approach of HPLC-coupled electrospray ionization mass spectrometry. Exposure to sodium azide (5 mM) increased the total amount of NAPE threefold over control levels; however, no alteration of the relative composition of NAPE species was detected. The anandamide precursor (20 : 4-NAPE) constituted only 0.1% of all NAPEs detected in the neurons. Total NAPE species in control cells amounted to 956-1,060 pmol/10(7) cells. Moreover, we detected the presence of an unknown NAPE species with molecular weight identical to 20 : 4-NAPE. This may suggest the presence of a putative stereoisomer of the anandamide precursor with at least one trans-configured double bond in the N-arachidonoyl moiety. These results show that with the present method, neuronal NAPE species can be identified and quantified with respect to N-acyl composition, including a trans-isomer of the anandamide precursor. The anandamide precursor is up-regulated to the same extent as other NAPEs upon neuronal injury.