Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [³H]serotonin, [³H]noradrenaline, or [³H]glutamate occurred. Serotonin, noradrenaline, and N -acetylaspartyl glutamate inhibited active transport of [³H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ-aminobutyric acid were shown here not to inhibit active transport of [³H]-acetylcholine. Noradrenaline was competitive with respect to [³H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [³H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.     

Metabolism of Deoxyuridine in Rabbit Brain

Abstract: The metabolism of [³H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [³H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [³H]deoxyuridine was metabolized to [³H]deoxyuridine phosphate, with subsequent conversion to [³H]thymidine phosphate, and ultimately [³H]DNA. The percentage of the [³H]deoxyuridine phosphorylated and subsequently converted into [³H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [³H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [³H]deoxyuridine to [³H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [³H]deoxyuridine, over 90% of the [³H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the ³H remaining in brain was phosphorylated and converted to [³H]thymidine phosphates; and 11 and 4%, respectively, of the ³H remaining in brain was converted to [³H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.     

Effects of Arachidonic Acid on Dopamine Synthesis, Spontaneous Release, and Uptake in Striatal Synaptosomes from the Rat

Abstract: Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [³H]dopamine ([³H]DA) continuously synthesized from [³H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [³H]H₂O formation (an index of [³H]tyrosine conversion into [³H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [³H]DA synthesis. In contrast to AA, arachidic acid, oleic acid, and the methyl ester of AA (all at 10⁻⁴ M ) did not modify [³H]DA release. The AA (3 × 10⁻⁵ M )-evoked release of [³H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [³H]DA uptake into synaptosomes, with a complete blockade observed at 10⁻⁴ M . However, AA (10⁻⁴ M ) still stimulated [³H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10⁻⁴ M )-evoked release of [³H]DA was not affected by protein kinase A inhibitors (H-89 or Rp -8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).     

Phosphatidylserine Enhancement of [3H]γ-Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes

Abstract: [³H] γ -Aminobutyric acid ([³H]GABA) binding to purified lipids was examined in an organic solvent-aqueous partition system. In addition, the [³H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium-dependent [³H]GABA uptake into synaptosomes isolated from rat whole brains. [³H]GABA was found to bind to all of the lipids studied in the organic solvent-aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [³H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [³H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μ m ). These results suggest that PS may play a role in the sodium-dependent GABA reuptake process in the presynaptic nerve end.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

Comparative Analysis of Different Approaches to Investigate Cell Kinetics

Abstract. The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [³H]-thymidine autoradiography ([³H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [³H]TdR LI and BrdU LI ( r _s= 0.90; P < 0.01), [³H]TdR LI and S phase ( r _s= 0.62; P < 0.01) and [³H]TdR LI and Ki67 ( r _s= 0.64; P < 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [³H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [³H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Pharmacology and Regional Distribution of the Binding of 6-[3H]Nitro-7-Sulphamoylbenzo[f]-Quinoxaline-2,3-Dione to Rat Brain

Abstract: 6-Nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [³H]NBQX binding to rat brain. The association rate of [³H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 m M unlabelled l -glutamate. [³H]NBQX bound to a single class of sites with K _D and B _max values of 47 n M and 2.6 pmol mg⁻¹ of protein, respectively. The rank order of inhibition of [³H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ≥ ( S )-5-fluorowillardiine ≥ AMPA ≫ l -glutamate. The chaotrope KSCN had no effect on the IC₅₀ value of unlabelled NBQX displacement of [³H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [³H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [³H]NBQX to coronal sections showed a distribution compatible with that of [³H]AMPA binding. These data indicate that [³H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.     

NEUROCHEMICAL PARAMETERS IN THE HYPERRESPONSIVE PHASE AFTER A SINGLE DOSE OF NEUROLEPTICS TO MICE

Abstract— Four days after a single dose of teflutixol (5 mg/kg i.p.), at which time mice are superresponsive to dopamine agonists, e.g. apomorphine, the specific binding of [³H]haloperidol, [³H]cis (Z)-flupenthixol, [³H]apomorphine, [³H]dopamine, [³H]propylbenzilylcholine mustard and [³H]GABA to striatal membranes in vitro is equal to that of saline-treated mice. Specific binding of [³H]haloperidol is also unchanged 3 days following a single dose of fluphenazine (5mg/kg i.p.) and 2 days following haloperidol (5 mg/kg i.p.), but slightly decreased 3 days following cis(Z)-flupenthixol (5 mg/kg i.p.).
The possibility that remaining neuroleptic or active metabolites could obscure a slight increase in dopamine receptor binding was rejected, since remaining amounts of [³H]teflutixol in the final binding assay 4 days after intraperitoneal injection of [³H]teflutixol (5 mg/kg) were too small to influence the binding of [³H]haloperidol in vitro .
It is concluded that the pharmacological superresponsiveness and the decrease in dopamine synthesis and release seen after the initial receptor blockade following a single dose of neuroleptic drugs in mice are nor accompanied by changes in dopamine, muscarine or GABAergic receptor characteristics in corpus striatum. The possibility that changes occur in a small number of functional operative dopamine receptors cannot be excluded, however.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

[3H]Xylamine Accumulation by Two Sympathetically Innervated Tissues

Abstract: The accumulation of [³H]xylamine ([ring-G-³H]- N -2'-chloroethyl- N -ethyl-2-methylbenzylamine; [³H]XYL) by rabbit thoracic aorta and rat vas deferens was studied in vitro . [³H]XYL uptake in both tissues saturated at the same concentrations and displayed the same time course as that for maximal inhibition of [³H]noradrenaline ([³H]NA) accumulation by XYL. Hydrolysis of [³H]XYL or coincubation with Na₂S₂O₃ reduced tissue accumulation of tritium by 80%. In aorta and vas deferens, about 45% and 65%, respectively, of the total [³H]XYL accumulation was blocked by 100 μmol/L desmethylimipramine (DMI), l -NA, or bretylium. In the absence of sodium ion, the uptake of [³H]XYL was reduced by approximately these same amounts. In both tissues nearly 70% of the [³H]XYL taken up was protein bound when measured as trichloroacetic acid-precipitated tritium. Of this radioactivity, the same proportion was sensitive to 100 μmol/L DMI or the absence of sodium ion as found for total tissue accumulation of [³H]XYL. Hydrolysis of [³H]XYL prior to incubation with vas deferens reduced the protein-bound tritium by more than 95%. The studies indicate that [³H]XYL interacts with NA uptake carrier in both rabbit aorta and rat vas deferens and that a substantial portion of the resulting protein-bound radioactivity is carrier-dependent.     

MELATONIN: DEACETYLATION TO 5-METHOXYTRYPTAMINE BY LIVER BUT NOT BRAIN ARYL ACYLAMIDASE

Abstract— Rat liver and brain slices were incubated in vitro with [³H]melatonin. Liver slices synthesized small amounts of [³H]5-methoxyindoleacetic acid ([³H]5-MIAA) along with other melatonin metabolites including 6-hydroxymelatonin. Pretreatment of animals prior to killing with the irreversible monoamine oxidase inhibitor pargyline allowed [³H]5-methoxytryptamine ([³H]5-MT) to be recovered from the incubation. No [³H]5-MIAA or [³H]5-MT could be detected in incubations with hypothalamic slices or following intraventrieular injection of [³H]melatonin. The possibility that the deacetylase aryl acylamidase was in part responsible for the deacetylation occurring in liver slices was examined. Liver aryl acylamidase was able to utilize [³H]melatonin as substrate to produce [³H]5-MT. Furthermore, the liver enzyme was inhibited by melatonin ( K_i. 1 m m ) when tested with the alternate substrate o -nitroacetanalide. Brain aryl acylamidase did not generate any detectable [³H]5-MT nor was it inhibited by melatonin. These results suggest that 5-MT is not formed in brain from melatonin although trace amounts of 5-MT in the periphery could be derived from this precursor.