Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices

Abstract: Using rat striatal slices, we examined the effect of cytidine on the conversion of [³H]choline to [³H]-phosphatidylcholine ([³H]PC), and on net syntheses of PC, phosphatidylethanolamine (PE), and phosphatidylserine, when media did or did not also contain choline, ethanolamine, or serine. Incubation of striatal slices with cytidine (50–500 µM) caused dose-dependent increases in intracellular cytidine and cytidine triphosphate (CTP) levels and in the rate of incorporation of [³H]choline into membrane [³H]PC. In pulse-chase experiments, cytidine (200 µM) also increased significantly the conversion of [³H]choline to [³H]PC during the chase period. When slices were incubated with this concentration of cytidine for 1 h, small (7%) but significant elevations were observed in the absolute contents (nmol/mg of protein) of membrane PC and PE (p < 0.05), but not phosphatidylserine, the synthesis of which is independent of cytidine-containing CTP. Concurrent exposure to cytidine (200 µM) and choline (10 µM) caused an additional significant increase (p < 0.05) in tissue PC levels beyond that produced by cytidine alone. Exposure to choline alone at a higher concentration (40 µM) increased the levels of all three membrane phospholipids (p < 0.01); the addition of cytidine, however, did not cause further increases. Concurrent exposure to cytidine (200 µM) and ethanolamine (20 µM) also caused significantly greater elevations (p < 0.05) in tissue PE levels than those caused by cytidine alone. In contrast, the addition of serine (500 µM) did not enhance cytidine's effects on any membrane phospholipid. Exposure to serine alone, however, like exposure to sufficient choline, increased levels of all three membrane phospholipids significantly (p < 0.01). These data show that exogenous cytidine, probably acting via CTP and the Kennedy cycle, can increase the synthesis and levels of membrane PC and PE in brain cells.     

Presence of Phospholipid-N-Methyltransferases and Base-Exchange Enzymes in Rat Central Nervous System Axolemma-Enriched Fractions

Rat CNS myelinated axons were fractionated by sucrose density gradient centrifugation with a zonal rotor. Fraction VI, obtained at 28-30% sucrose, appeared, on the basis of the presence of related marker enzymes, to be enriched in axolemma. Phospholipid-N-methyltransferases (PMTs) and base-exchange enzymes were associated with fraction VI. PMT activity was significantly stimulated by the addition of either phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine but the PMT activity of the homogenate or the myelinated axons was unresponsive. Recoveries of the ethanolamine, serine, and choline base-exchange activities were 14.4%, 13.8%, and 3.4%, respectively, of that present in the myelinated axons. The myelin-rich fraction obtained simultaneously seems contaminated with other membrane fractions.     

Sidedness of Phosphatidylcholine-Synthesizing Enzymes in Rat Brain Microsomal Vesicles

The sidedness of CDP-choline:1,2-diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base-exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury-dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP-choline:1,2-diradylglycerol choline phosphotransferase and the choline base-exchange activity are localized on the outer surface of rat brain microsomal vesicles.     

Ethanolamine Kinase Activity in Purified Myelin of Rat Brain

Highly purified rat brain myelin showed a significant level of ethanolamine kinase, amounting to 17% of the specific activity of whole brain homogenate. This kinase level in myelin was an order of magnitude higher than that of lactate dehydrogenase, a marker for cytosol. Subcellular distribution studies revealed that in addition to myelin, this kinase was present in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. The possibility that myelin activity resulted from adsorption of the soluble enzyme was unlikely since activity was retained in myelin that had been washed with buffered sodium chloride or taurocholate. Mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin. Kinetic studies indicated similar Km values for ethanolamine in the microsomal, cytosolic, and myelin fractions but a significantly lower apparent Km for ATP in myelin. This and other differences suggested the possible existence of isozymes. Establishment of the presence of this kinase completes the list of phospholipid synthesizing enzymes needed to synthesize phosphatidylethanolamine from diacylglycerol within the myelin membrane.     

Release of Ethanolamine, but Not of Serine or Choline, in Rat Pontine Nuclei on Stimulation of Afferents from the Cortex, In Vivo

Release of ethanolamine, serine, and choline in rat pontine nuclei on electrical stimulation of afferents from the cortex was investigated using in vivo push-pull cannula techniques. Ethanolamine was determined by using gas chromatographic techniques; serine was measured with a HPLC system; and choline was assayed with a luminescence method. Resting elution rates of ethanolamine, serine, and choline were 50.8 +/- 8.4, 34.8 +/- 12.6, and 1.16 +/- 0.20 pmol/5 min, respectively. Stimulation of the cortico-pontine tract evoked a highly significant 3.4-fold increase in release of ethanolamine, whereas serine and choline release was unaffected. Reactions in membrane phospholipids are most likely involved in the stimulation-dependent release of ethanolamine and special consideration was given to base-exchange reactions. Alternatively, a release from intracellular, possibly synaptic stores cannot be excluded.     

Concomitant Increases in the Levels of Choline and Free Fatty Acids in Rat Brain: Evidence Supporting the Seizure-Induced Hydrolysis of Phosphatidylcholine

The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 micrograms/kg, s.c.), at a dose that produced toxic effects, increased the levels of both free fatty acids (175-250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman-induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman-induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant-mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.     

Effect of Unilateral Decortication on Choline Acetyltransferase Activity in the Nucleus Basalis and Other Areas of the Rat Brain

Acetyl-coenzyme A: choline O-acetyltransferase (EC 2.3.1.6) (ChAT) enzyme activity was measured in the nucleus basalis and other microscopically identified brain areas at various times after unilateral cortical lesions were made in the rat. Initially, a significant decrease in ChAT activity was detected in the nucleus basalis ipsilateral to the lesion. However, after 120 days ChAT activity had apparently recovered, as levels of the enzyme at that time were not significantly different from control values. No changes in ChAT activity could be detected in any of the other brain areas similarly studied. The significance of these findings and their relationship to the morphological changes seen in neurones of the nucleus basalis after cortical lesions are discussed.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Acetylation of Choline and Homocholine by Membrane-Bound Choline-O-Acetyltransferase in Mouse Forebrain Nerve Endings

Abstract: The choline analog homocholine is not acetylated in vitro by choline- O -acetyltransferase (ChAT, EC 2.3.1.6), which is solubilized by 100 mM-sodium phosphate buffer washes of a crude vesicular fraction of mouse forebrain. However, both homocholine and choline are acetylated by a form of ChAT which is nonionically associated with a subcellular fraction of mouse forebrain containing membrane-associated organelles and occluded acetylcho-line (P₄). Acetylation of homocholine by membrane-associated ChAT is saturable. 4-(1-Naphthylvinyl)pyridine (NVP) inhibits the acetylation of both choline (60%) and homocholine (40%) by membrane-associated ChAT but reduces the acetylation of choline alone by soluble ChAT (76%). Choline and homocholine serve as competitive alternative substrates for the same membrane-associated ChAT, whereas homocholine acts only as a competitive inhibitor of choline acetylation by soluble ChAT. Acetylhomocholine competitively inhibits the acetylation of choline by both soluble and membrane-associated ChAT more dramatically than does the natural end product, acetylcholine.     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

The Presence of (+)-S-Adenosyl-l-Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N-Methyltransferase Activity

Abstract: (+)-S-Adenosyl- l -methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by ¹H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N -methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N -methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.     

Noncorrelation of Choline Kinase or ATP Citrate Lyase with Cholinergic Activity in Rat Brain

Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.     

Effect of Low pH on Glutamate Uptake and Release in Isolated Presynaptic Endings from Rat Brain

The effect of acidification of the incubation medium on the membrane potential and glutamate uptake and release was studied in isolated presynaptic neuronal endings (synaptosomes) from rat brain. Using the fluorescent probe diS-C₃-(5), a rapid depolarization of plasma membrane was detected at pH 6.0, most probably as a result of the inhibition of the sodium pump and potassium channel blockade. The membrane potential decrease did not result in increase of basal efflux of glutamate. Glutamate release following K⁺-induced depolarization was decreased upon lowering pH to 6.0. Acidosis inhibited mainly calcium-dependent (vesicular) release of glutamate and did not significantly reduce [¹⁴C]glutamate uptake. This inhibition of glutamate release but not of glutamate uptake may be a mechanism of the protective effect of acidosis during brain ischemia.     

Effect of Serine and Ethanolamine Administration on Phospholipid-Related Compounds and Neurotransmitter Amino Acids in the Rabbit Hippocampus

Abstract: The report concerns mechanisms for the increase of extracellular levels of ethanolamine and phosphoethanolamine in CNS regions, such as the hippocampus, in transient brain ischemia, hypoglycemia, seizures, etc. l -Serine (2.5–10 m M ), d -serine (10 m M ), or ethanolamine (10 m M ) was administered for 20 min via a microdialysis tubing to the hippocampus of unanesthetized rabbits. The concentrations of primary amines were determined in the dialysates. When levels were elevated 10–100 times in the extracellular fluid, l -serine caused a dose-dependent increase of the concentration of extracellular ethanolamine. Ethanolamine caused a corresponding, although somewhat smaller, increase in serine levels. Furthermore, l -serine also induced an increased concentration of phosphoethanolamine that was delayed in time relative to the peak of ethanolamine. d -Serine was as effective as l -serine in raising ethanolamine levels but had no effect on phosphoethanolamine. Ethanolamine, but not l -serine, also increased extracellular glutamate/aspartate levels in an MK-801-dependent fashion. A similar effect, but delayed in time, was observed with d -serine. These effects were inhibited by MK-801. The concentrations of other amino acids were not significantly affected. The characteristics of the effects are suggestive of base exchange reactions between serine and ethanolamine and between ethanolamine and serine glycerophospholipids, respectively, in neuronal plasma membranes.     

Analytical Subcellular Fractionation of Rat Cortex: Resolution of Serotonergic Nerve Endings and Receptors

Abstract: An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two-step procedure involving a differential centrifuga-tion using the five-fraction scheme and an isopycnic cen-trifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S₂ receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mito-chondrial (M and L) fractions. After equilibration in density gradients, serotonin S₂ receptors revealed two peaks, which were similarly affected after treatment with ami-triptyline and/or yohimbine. There is no evidence to suggest that serotonin S₂ receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S₂ receptors and 5′-nucleoti-dase are very similar, but differ from that of UDP-galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5-HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5′-nucleotidase, and receptor binding), is rapid and clearly differentiates pre-and postsynaptic constituents.     

Acetylation of Homocholine by Rat Brain: Subcellular Distribution of Acetylhomocholine and Studies on the Ability of Homocholine to Serve as Substrate for Choline Acetyltransferase In Situ and In Vitro

Abstract: In situ acetylation of homocholine by slices of rat cerebral cortex was about 34% of the in situ acetylation of choline. Acetylhomocholine synthesized by the cerebral cortical slices was distributed in the same subcellular fractions as was acetylcholine (ACh), although the relative distribution of acetylhomocholine and ACh between nerve-ending-free and nerve-ending-bound stores was different. Cerebellar slices acetylated homocholine <10% as well as did cerebral cortical slices. In vitro , choline acetyltransferase (ChAT; EC 2.3.1.1.6) either partially purified from whole rat brain, solubilized from lysed synaptosomes, or in a synaptosomal membrane-associated form, did not acetylate homocholine at an appreciable rate. Under conditions of alkaline pH, an appreciable in vitro rate of homocholine acetylation by preparations of lysed synaptosomes was detected. However, analysis of this acetylation showed it not to be the result of ChAT catalysis and unlikely to occur by the same mechanism as that responsible for acetylation of homocholine in situ : the acetylation was not inhibited by ChAT inhibitors and occurred equally in the presence of preparations of lysed cerebral cortical or cerebellar synaptosomes. It is concluded that in situ acetylation of homocholine is probably catalyzed by ChAT and that acetylhomocholine is subsequently stored in the same subcellular sites as is ACh; the inability to detect ChAT-catalyzed acetylation of homocholine in vitro might arise as an artefact of the procedures employed in isolation of the enzyme.     

Purification and Some Characteristics of an ACTH-Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane-bound protein (B-50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X-100 in 75 mM-KCl. Using column chromatography over DEAE-cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B-50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B-50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B-50 after 60 min. The inhibition by ACTH_{1_24} of the B-50 protein kinase in ASP 55–80 was dose-dependent; the half-maximal effective concentration was 5 × 10⁻⁶ M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor-phins had no effect on the B-50 protein kinase. The B-50 protein kinase required both magnesium and calcium for optimal activity. Using two-dimensional electrophoresis on polyacrylamide slab gels the B-50 protein kinase and the B-50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B-50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B-50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B-50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.     

Concentrations of Tryptoline and Methtryptoline in Rat Brain

Combined gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5-hydroxytryptoline, and 5-hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptoline were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neuroanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5-hydroxytryptoline and 5-hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated-5-hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5-hydroxytryptoline and 5-hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.     

Further Characterization of Phasic Calcium Influx in Rat Cerebrocortical Synaptosomes: Inferences Regarding Calcium Channel Type(s) in Nerve Endings

Under conditions minimizing the contribution of Na+/Ca2+ exchange to calcium entry in synaptosomes, the K+ depolarization-dependent calcium influx (JCa) is a single exponential function of time. JCa activates and slowly inactivates at membrane potentials positive to -50 mV, a result indicating the involvement of moderate voltage-activating, slowly inactivating calcium channels. Calcium channels in synaptosomes are characterized by stronger sensitivity to blockage by Cd2+ than Co2+, insensitivity to dihydropyridine calcium antagonists or the agonist Bay K 8644, and weak, partial sensitivity to the peptide toxin omega-conotoxin GVIA. These characteristics suggest that voltage-sensitive calcium channels in rat cerebrocortical synaptosomes are dissimilar from the somatic T, N, or L channel types. JCa is not affected by treatment of synaptosomes with the adenylate cyclase activator forskolin, the membrane permeant dibutyryl-cyclic AMP, or the kinase C activator phorbol 12-myristate 13-acetate diester, results suggesting that calcium channels in synaptosomes are not directly modulated by protein kinase A- or C-mediated phosphorylation.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.