MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

TRIETHYLTIN AND THE INCORPORATION OF [33P] PHOSPHATE INTO RAT BRAIN PHOSPHOLIPIDS

By the use of quantitative thin-layer chromatography, it has been shown that triethyltin sulphate 7.5 mglkg body wt. reduces the incorporation of ⁸⁸P₁ into rat brain phospholipids, especially lecithin, when the animals are kept at an environmental temperature of 20°. Triethyltin at this dose also reduces the body temperature by approximately 6°. When the body temperature of the triethyltin-treated animals is maintained at a normal level by placing them in an environmental temperature of 33°, no significant reduction in the incorporation of ³²P₁ into any of the phospholipids is observed.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

BIOSYNTHESIS OF LACTOSYLCERAMIDE BY RAT BRAIN PREPARATIONS AND COMPARISON WITH THE FORMATION OF GANGLIOSIDE GM1 AND PSYCHOSINE DURING DEVELOPMENT

—This report deals with some properties of the enzyme from rat brain, which catalyses in vitro the formation of lactosylceramide, a probable precursor of the gangliosides, from UDP-galactose and glucosylceramide. The enzyme is present in microsomes, mitochondria and synaptosomes, the latter having the highest specific activity, and appears to be firmly bound to the membranes of these particles. The enzymic activity is optimal at pH 6·8 and requires the presence of Mn²⁺. Lactosylceramide, the product of the reaction, was tentatively identified by its elution pattern from a Florisil column, its resistance to mild alkaline hydrolysis, thin-layer co-chromatography with authentic standards in six solvent systems, and location of the radioactivity in the galactose portion of the product obtained after incubation of labelled UDP-galactose with unlabelled glucosylceramide and in the glucose portion after incubation of glucose-labelled glucosylceramide with unlabelled UDP-galactose. The activity of this enzyme, per unit of brain weight, was found to be highest at birth and to decrease gradually thereafter. A similar age distribution was observed for another galactosyltransferase, the one which catalyses the formation of ganglioside G_M1 from Tay-Sachs’ganglioside. In contrast, the activity of a third galactosyltransferase, which catalyses the formation of psychosine, and thus possibly is related to the increase of cerebrosides during myelination, is negligible during the first week and maximal about 20 days after birth.     

UPTAKE OF ACETYLCHOLINE AND CHOLINE INTO RAT BRAIN CORTICAL SLICES AND SYNAPTOSOMES AS RELATED TO 32Pi, INCORPORATION INTO THEIR PHOSPHOLIPIDS

—The role of ACh-stimulated ³²P_i incorporation into the phospholipids of rat cerebral cortex slices and isolated nerve endings (synaptosomes) has been studied. ACh stimulation is not connected with any carrier-mediated uptake of ACh. Such uptake may occur in slices in the presence of the anticholinesterase Sarin but barely in the presence of eserine. Regardless of the nature of the anticholinesterase used, rat cerebral cortex synaptosomes that respire and show high and low affinity choline uptake do not accumulate ACh against a concentration gradient. At exogenous ACh concentrations of 10^–5m and above, some ACh enters the synaptosomes by diffusion and significantly stimulates ³²P_i incorporation into phosphatidic acid. It is discussed whether, in isolated nerve endings, an increase in cytoplasmic ACh concentration due to diffusion may induce vesicle turnover to keep a balance between ‘free’ and bound ACh or if a presynaptic ACh receptor is responsible for the observed changes in phosphatidic acid. The distribution of accumulated radioactivity derived from exogenous choline and ACh respectively between ACh, choline, phosphorylcholine and betaine has been studied in slices and isolated nerve endings.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

Regional Distribution of Prostaglandins D2, E2, and F2α and Related Enzymes in Postmortem Human Brain

Abstract: The presence of prostaglandins D₂, E₂, and F_2α was demonstrated and their contents measured in various regions of postmortem human brain, pineal body, and pituitary by using specific radioimmunoassays and gas chromatography-mass spectrometry. The three prostaglandins were widely distributed in similar concentrations ranging from several hundred pg/g wet weight to about 40 ng/g wet weight. Prostaglandins D₂ and E₂ showed consistent and similar regional distributions in all six brains tested; amounts were high in pineal body, pituitary, olfactory bulb, and hypothalamus. On the other hand, prostaglandin F_2α was distributed more evenly. Prosta- glandin D synthetase and prostaglandin E synthetase activities were found in cerebrum homogenate from a single subject and were recovered from the 100,000 × g supernatant. The presence of 1 m M glutathione, reduced form, markedly stimulated the activity of prostaglandin E synthetase, but did not affect prostaglandin D synthetase activity. Activity of 15-hydroxyprostaglandin dehydrogenase was found in the cerebrum homogenate and was partially purified. This enzyme required NADP as a cofactor and copurified with prostaglandin E 9-ketoreductase.     

INCORPORATION OF d-[3H]GLUCOSAMINE INTO THE SIALOGLYCOPROTEIN GP-350 OF ADULT RAT BRAIN

—The incorporation of d -[³H]glucosamine into the nervous specific sialoglycoprotein GP-350 has been studied in adult rat brain. Both the 100,000 g supernatant fluid and the 12,500 g pellet were used for the investigation, since GP-350 could only be detected in the soluble cell fraction (Van Nieuw Amerongen et al., 1972) and in the synaptosomal membranes, sedimenting in the crude mitochondrial fraction (Van Nieuw Amerongen & Roukema , 1973, 1974). GP-350 was separated from the other proteins by polyacrylamide gel electrophoresis at pH 7.5 and the incorporation of radioactivity into GP-350 was measured at different time intervals, ranging from 1 to 96 h after the administration of the radioisotope. The maximal incorporation of radioactivity into the soluble GP-350 was obtained after about 2 h and into the membrane-bound GP-350 after about 3 h. After 2 h there is a very rapid decrease of the radioactivity of GP-350 from the soluble cell fraction (up to 70 per cent within 2 h). Thereafter the disappearance is more gradual and of the same order as that found for the membrane-bound fraction of GP-350. The half-life of the soluble GP-350 was estimated to be 19 h and for the membrane-bound GP-350 a value of 18 h was calculated. Compared to the total pool of brain (glyco) proteins and specific nervous (glyco) proteins GP-350 has a very rapid turnover. The rapid initial decrease of the radioactivity from the soluble GP-350 may be interpreted in terms of a rapid transport of the newly-synthesized GP-350 from the cytoplasma of the perikaryon to the membranes of the synaptic region by an axoplasmic flow.     

GLYCOPROTEINS IN BRAIN TISSUE OF THE O-VARIANT OF GM2 GANGLIOSIDOSIS

Abstract— The dialysableglycopeptide preparation recovered from the glycoproteins in cerebral gray matter of a case of the O-variant form of G_M2 gangliosidosis contained four fold more N -acetylglucosamine and mannose than a similar preparation from normal gray matter. In the O-variant form of G_M2 gangliosidosis, the enzymes β - N -acetylhexosaminidases A and B are missing. A three- and four-fold elevation, respectively, of N -acetylglucosamine and mannose in the dialysable glycopeptide preparation from a case of Tay-Sachs disease (B-variant form of G_M2 gangliosidosis) was noted. The B-variant lacks hexosaminidase A but has ample supplies of hexosaminidase B. The brain level of glycosaminoglycans was not affected in the O- and B-variant forms of G_M2 gangliosidosis.     

DECREASED RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO AFTER EXPOSURE TO ATMOSPHERES CONTAINING HIGH CONCENTRATIONS OF CO2

—(1) The effects of exposure of rats to increased atmospheric concentrations of CO₂ on brain metabolism in vivo were studied. (2) After 2·5 min exposure to an atmosphere of 20% CO₂, the rate of glucose utilization by brain decreased from 0·61 μmol/min per g to 0·32 μmol/min per g and remained between 0·3 and 0·4 μmol/min per g for 60 min, the longest interval studied. O₂ utilization, calculated from the arteriovenous difference of O₂ across the brain and blood flow, was 3·5 μmol/min per g in controls and was 4·7 μmol/min per g after 5 min in the 20% CO₂ atmosphere. (3) The concentrations of glucose, glucose 6-phosphate and aspartate were increased during the first 10 min of CO₂ exposure whereas the concentrations of other glycolytic intermediates, tricarboxylic acid cycle intermediates and glutamate were decreased. The amount of endogenous substrate which disappeared during the first 10 min was sufficient, if used to supplement glucose as a fuel, to maintain the O₂ consumption at, or slightly above, the control level. Glutamate and lactate were quantitatively the most important energy sources. (4) The mechanism whereby‘CO₂ decreased the rate of glucose utilization is uncertain. The initial rise in glucose 6-phosphate and fall in fructose 1,6-diphosphate concentrations suggested that an inhibition of phosphofructokinase was responsible. However, after 60 min in 20% CO₂, the concentrations of both of these metabolites returned to normal while the rate of glucose utilization remained depressed.     

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

Abstract— Radioactive acetylcholine ([¹⁴C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[¹⁴C]ACh medium, apparently by a passive diffusion process at concentrations > 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm -[¹⁴C]ACh and about constant, 27%, for the range 5-50mm -[¹⁴C]ACh. All its ACh is released on homogenization with 0.1 n -perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [¹⁴C]ACh. Deprivation in the incubation media of glucose or Na⁺ or Ca²⁺. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na⁺, Ca²⁺ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na⁺ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.     

EFFECTS OF SPREADING CORTICAL DEPRESSION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEINS OF RAT BRAIN

Abstract— Incorporation of dl -[1-¹⁴C]leucine into proteins of the cerebral cortex of the rat was measured during spreading cortical depression (CSD) evoked by a single topical application of 25% (w/v) KCI. Maximal inhibition (42 per cent) of the rate of incorporation occurred 1 hr after application of KCI. Spreading depression of 2–3 hr duration was associated with 22 per cent and 13 per cent decreases, respectively, of incorporation of labelled leucine. Specific activity of the free pool leucine was not decreased during CSD but appeared to be higher than controls at 20 min after initiation of CSD. The specific activity of the total free pool amino acids was also increased at 10, 20, 60 and 120 min after application of KCI.
The inhibitory effect of CSD on incorporation of leucine into proteins was uniformly distributed among the crude mitochondrial, microsomal and soluble subcellular fractions from brains of adult animals, while in fractions from 25-day old animals there appeared to be relatively more inhibition in the crude mitochondrial fraction.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

THE EFFECT OF N-METHYL-4-PIPERIDYL-DIPHENYL GLYCOLATE ON THE INCORPORATION OF 32P INTO PHOSPHOLIPIDS FROM RAT BRAIN CORTEX SLICES AND ITS SUBCELLULAR LOCALIZATION

—N-Methy-4-piperidy1-diphenyl glycolate (N-methy1-4-piperidy1 benzilate), an anticholinergic drug, was shown to stimulate ³²P-incorporation into total phospholipids of rat brain cortex slices. Analysis of the total phospholipids showed stimulation in phosphatidic acid, phosphatidylinositol and phosphatidylethanolamine. Stimulated ³²P-incorpora-tion was accompanied by a decrease in the Qo₂ (μ1 O₂/mg dry tissue) value. The effects of the drug were compared with those of some other CNS-active drugs known to interfere with the ACh content of brain; tremorine, oxotremorine and atropine; and in the presence of eserine, with that of the neurotransmitter acetylcholine. Increase of the outer K+-concentration resulted in increased Qo₂ and ³²P-incorporation into the slices. The effect of the glycolate and perhaps that of atropine tended to increase in this medium. Subcellular fractionation of slices showed that the glycolate stimulated ³²P-incorporation occurred mainly in the nerve end fraction. The total amount of the individual phospholipids was not changed, but the specific activity had significantly increased in phosphatidic acid and phosphatidylinositol. The possibility that glycolate-induced stimulated ³²P-incorporation into nerve end phospholipids is due to increased glycolysis is discussed.