Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [³H]glycerol, [³H]myristate, [³H]choline or [³H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [³H]glycerol or [³H]myristate were stimulated by vasopressin, no significant decrease in the labelling of phosphatidylcholine was detected, but the labelling of phosphatidic acid was elevated. However, the labellings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [³H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells.Abbreviations BHK-21 cells baby hamster kidney-21 cells     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

(14C) acetylcholine synthesis by cortex slices of rat brain

Abstract—

• 1 A procedure has been developed to measure ACh synthesis from [¹⁴C]-precursors. As little as 10^?9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG-50, precipitation and recrystallization of ACh chloroaurate.
• 2 [U^?14C]glucose and [2^?14C]pyruvate formed similar amounts of [¹⁴C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [¹⁴C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden-Meyerhof pathway.
• 3 The amount of ACh detected by bioassay after incubation of cortex slices with [U^?14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose.
• 4 [¹⁴C]choline, either methyl or chain labelled, formed [¹⁴C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose.
• 5 When both [U^?14C]glucose and [¹⁴C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10^?4m -hemicholinium-3 inhibited ACh formation by more than 90 per cent from either [U-¹⁴C]glucose or [Me-¹⁴C]choline.
• 6 Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [¹⁴C]ACh within the slices which reached a maximum during the first hour of incubation. [¹⁴C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm -KCI markedly increased the formation of [¹⁴C]ACh found in the incubation medium. Decreased amounts of [¹⁴C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.

     

BIOSYNTHESIS OF RAT BRAIN PHOSPHATIDYLCHOLINES FROM INTRACEREBRALLY INJECTED CHOLINE1

Abstract— [Me-³H] Choline was injected intracerebrally into male rats and the brains immediately removed by particular procedures at regular intervals over the first 1200 s. The incorporation of radioactivity into brain phosphorylcholine, CDP-choline and phosphatidylcholines was examined and quantitated, in order to investigate the relative roles of net synthesis and base-exchange reactions for choline incorporation into lipid. The molecular subspecies of phosphatidylcholines were also examined after isotope administration. Phosphorylcholine, CDP-choline and phosphatidylcholines all became labelled as early as 5 s after the administration of labelled choline. The time course of incorporation of choline into brain lipid is biphasic with two flex points at about 20 and 120 s from the injection. The specific radioactivity of different phosphatidylcholines appears to be different at early and later intervals from injection. The suggestion is made that the base-exchange pathway for choline incorporation into lipid might be operative in vivo in early periods after administration.     

Phosphatidylcholine Metabolism in Nuclei of Phorbol Ester-Activated LA-N-1 Neuroblastoma Cells

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuro-blastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [³H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [³H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPS. The stimulation of [³H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.     

Physiology of a choline-requiring strain of Torulopsis pintolopesii

Summary A choline-requiring strain of Torulopsis pintolopesii when growing on choline-methyl-¹⁴C or choline-methyl-³H excretes the radioactivity incorporated in the first 24–48 h of incubation up to ca. 85–95% of the radioactivity added at the beginning of the incubation. The addition of non-radioactive methionine did not interfere with the uptake and excretion of radioactivity from choline-Me-¹⁴C. Radioactive methyl group of methionine previously incorporated by growing cells of T. pintolopesii on varying concentrations of choline was not excreted in appreciable amounts. No evidence was obtained for the oxidation of choline to betaine, degradation to trimethylamine, or net incorporation of labelled choline into lecithin. The occurrence of a new pathway for the utilization of choline in yeasts is suggested. The requirement of choline by T. pintolopesii is explained tentatively by the formation and excretion of a compound containing the carbon and hydrogen atoms from the methyl groups of choline and whose chemical structure still under study, may comprise a heteroside containing mannitol as the polyhydroxylated moiety.     

ANALYSIS OF THE PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE BY CORTICAL SLICES FROM RAT BRAIN WITH THE AID OF TWO DIFFERENT LABELLED PRECURSORS

—The release of newly synthesized acetylcholine (ACh) by cortical slices from rat brain in the presence of 25 mm -KCl was studied. The slices were incubated for 5 min in a medium containing both [2-¹⁴C]pyruvate and choline labelled with 3 deuterium atoms (choline-d₃) in order to label at the same time the acetyl moiety and the choline moiety of ACh. The non-labelled ACh and the ACh-d₃ were measured by pyrolysis-gas chromatography/mass spectrometry, and the [^I4C]ACh by liquid scintillation counting. It was found that the newly formed [⁴C]ACh as well as the newly formed ACh-d₃ had a more than 2.5 times greater probability of being released than the preformed non-labelled ACh. These findings strongly suggest that it is not simply the ACh synthesized immediately inside the nerve ending membrane from incoming undiluted labelled choline, which is preferentially released, but that all newly formed ACh has a greater probability of being released than preformed ACh. No preferential release of newly formed ACh was observed when the incubation medium contained 5.6 mm -pyruvate instead of 10 mm -glucose + 0.6 mm -pyruvate. The cause of this difference remains unexplained.     

Acetylcholine turnover estimation in brain by gas chromatography-mass spectrometry

A gas chromatograph/quadrupole mass spectrometer system has been employed to estimate the turnover of acetylcholine in mouse brain $\text{in vivo}$ following a pulse intravenous injection of choline, using discrete deuterium labelled variants of choline and acetylcholine as tracer and internal standards. There appear to be two functional pools with turnover rates of 1.4 and 7.9 nmol gm^?1min^?1.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

Exogenous utilization of 14C sugars and 14C proline as carbon source for lipid biosynthesis in the germinating Crotalaria juncea L. pollen

Abstract

Of the ¹⁴C labelled sugars (sucrose, glucose and fructose) and ¹⁴C proline, the incorporation of label into different lipid types was least from proline while sucrose was the preferred precursor over glucose or fructose. High incorporation into phosphatidyl inositol with ¹⁴C sucrose suggested that this phosphatide besides being a membrane component served possibly as the inositol storage component. High incorporation of label into phosphatidyl choline also suggested synthesis of new membranes during pollen tube growth.     

Insulin stimulates turnover of phosphatidylcholine in rat adipocytes

The present study investigated the effect of insulin on phosphatidylcholine turnover in rat adipocytes labelled to equilibrium with [¹⁴C]-choline. Insulin induced a rapid turnover of this major phospholipid that was maximal by 1 min and transient in nature. Following a 1 min stimulation of the cells with insulin at a maximally effective concentration (7 nM), a 4–6% decrease in the percentage of total cellular choline associated with this phospholipid was observed. This reflected a significant transient increase in the percentage of total cellular choline associated with phosphorylcholine, which together with diacylglycerol are the phospholipase C cleavage products of phosphatidylcholine. These effects were observed over a physiological range of insulin concentrations. No effect of insulin on any other choline phospholipid or metabolite (sphingomyelin, lysophophatidylcholine, glycerophosphocholine or choline) was seen. These results suggest that insulin stimulates a phospholipase C-mediated turnover of phosphatidylcholine in rat adipocytes. The rapid nature of this turnover suggests a potential role in signal transduction.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

The effect of lidocaine on [³H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3·2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0·0005) or 55 per cent (n = 10; p < 0·0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [³H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until ³H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.     

IMPAIRED SYNTHESIS OF ACETYLCHOLINE IN BRAIN ACCOMPANYING MILD HYPOXIA AND HYPOGLYCEMIA

Abstract— Lowering the concentration of oxygen or of glucose to which mouse and rat brains were exposed impaired the synthesis of acetylcholine from labelled precursors in vivo. Histotoxic hypoxia induced with KCN or anemic hypoxia induced with NaNO₂ (to oxidize hemoglobin to methemoglobin) reduced incorporation of [²H₄]choline into acetylcholine. This change in acetylcholine metabolism occurred with doses of KCN or NaNO₂ which did not alter the concentrations of ATP or ADP or the adenylate energy charge. Hypoglycemia induced by large doses of insulin also reduced the incorporation of [²H₄]choline into acetylcholine. Both hypoxia and hypoglycemia increased the concentration of choline in the brain. The specific activity of choline did not decrease in hypoxia; it did not decrease enough in hypoglycemia to explain the reduced incorporation of [²H₄]choline into acetylcholine. Pretreatment with the cholinesterase inhibitor physostigmine delayed the onset of both seizures and death in mice after induction of hypoxia by large doses of NaNO₂. Pretreatment with physostigmine also decreased the number of mice dying within 3 h after the induction of hypoglycemia with large doses of insulin. These observations suggest that the effects of hypoxia and hypoglycemia interfere with the synthesis of a critical pool of acetylcholine. The incorporation of labelled precursors into acetylcholine related linearly to both the cytoplasmic redox state (NAD/NADH ratio) and to the NAD/NADH potential across the mitochondrial membrane. The redox potential of NAD/NADH in the cytoplasm was calculated from the [pyruvate]/[lactate] equilibrium and the redox potential of NAD/NADH in the mitochondria from the [NH4][2-oxoglutar-ate]/[glutamate] equilibrium. The potential across the mitochondrial membrane was calculated from the difference. These observations indicate that carbohydrate oxidation is one of the factors on which the synthesis of the neurotransmitter acetylcholine depends closely in mouse and rat brain.     

Digestion of phosphatidylcholines,absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphati-dylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1-[1-¹⁴C]acyl- and 1-[³H] alkyl-lysophosphatidylcholine, [1-¹⁴C]oleic acid, [2-¹⁴C]glycerol, phosphoryl[methyl-¹⁴C]choline, and [methyl-¹⁴C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A₂, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and P_i, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoalkylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine. Arch. Insect Biochem. Physiol. 36:273–293, 1997. © 1997 Wiley-Liss, Inc.     

Homologous and heterologous cell coupling in mammalian ovarian follicles

Movement of markers derived from ³H-labelled choline through the intact membrana granulosa has been quantified using scintillation counting. This passage is most readily explained in terms of junctional transfer and may be detected across in excess of 400 cell diameters. Whereas gonadotropins reduce the movement of choline between cumulus cells and the oocyte, that between somatic cells is unaffected. Both CO₂ and the calcium-transporting ionophore A23187 markedly reduce movement of labelled components and the reported effects of CO₂ and Ca²⁺ support the conclusions that the gradients are a result of junctional transfer. In addition, morphological examination of CO₂-treated follicles shows a loss of gap junctions.     

LACK OF PHOSPHOLIPID TRANSPORT MECHANISMS IN CELL MEMBRANES OF THE CNS1

The possible transport role of phospholipid-protein complexes, present in the cell supernatant of rat brain was investigated using labelled choline as precursor of phosphatidyl choline. Results obtained after the intracranial injection of choline gave no indication of a sequence of events compatible with a transport of phospholipid molecules from the possible site of synthesis (microsomes) to the supernatant and subsequently to myelin. Chase experiments using rat brain slices incubated in vitro with radioactive choline agreed well with the above mentioned results. Contrariwise, when Na³⁵₂SO₄ was used as precursor, the results clearly indicated that synthesis of sulphatides takes place in microsomes, followed by transfer of the radioactive lipid to sulphatide-containing lipoproteins in the supernatant and finally to myelin. Results presented in this paper seem to give further support to the idea that other subcellular fractions, besides microsomes, can autonomously synthesize part of their own provision of phospholipids. Possible reasons which might explain the marked differences between the mechanisms of addition of phospholipids and sulphatides to myelin are discussed in relation to results obtained by other investigators.     

Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes

Abstract— The calcium-dependent incorporation of l -[3-³H]serine and [1,2-¹⁴C]ethanol-amine into the phospholipid of isolated subcellular fractions from chick brain was studied and the properties of incorporation were examined. The microsomal fraction was found to possess the highest rate of incorporation and was able to convert under optimal conditions about 120 nmol of labelled serine and 220 nmol of ethanolamine/g of fresh brain microsomes/h. The requirement for Ca²⁺ ion appeared to be absolute. Mg²⁺ ion caused a gradual reduction in the existing enzymic activity, only when pre-incubated with microsomes and labelled bases before adding Ca²⁺ ion. The incorporation of serine and ethanolamine was actively inhibited by Hg²⁺, Co²⁺, Cu²⁺ and Mn²⁺ ions, and was abolished by ethylenediamine tetra-acetate treatment. Ethanolamine, but not choline, inositol or carnitine, competitively inhibited serine incorporation, while d -serine had slight effect. Conversely, l -serine inhibited competitively the incorporation of ethanolamine. The greater part of the incorporated serine (85 per cent) was localized in microsomal phosphatidylserine, while a small percentage was found in phosphatidylethanolamine. Similarly, 90 per cent of the incorporated ethanolamine was confined to phosphatidylethanolamine and a small percentage was found in the plasmalogen derivative. The mechanism of serine and ethanolamine incorporation was investigated. The results are compared with those published for similar mammalian and non-mammalian systems.     

EFFECTS OF RETINAL ABLATION ON UPTAKE OF GLUTAMATE, GLYCINE, GABA, PROLINE AND CHOLINE IN PIGEON TECTUM

—The effect of retinal ablation on the high and low affinity uptake of choline, GABA, glutamate, glycine and proline into the crude mitochondrial fraction of the pigeon optic tectum was studied. After 4–8 weeks of degeneration the uptake for glutamate, and to a lesser degree the uptake for GABA, at both the high and the low affinity substrate concentration, decreased. In contrast, the uptake of glycine increased. Kinetic analysis showed that the reduced uptake of glutamate was due to a decrease in the V_max. By intraocular injection of [³H]proline the retinal endings in the optic tectum were labelled with the fast axoplasmic transport fraction. Optic terminals labelled in this way have the same sedimentation characteristics in continous sucrose gradients as the particles accumulating giutamate in the uptake assay.