Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo

The incorporation of [7-³H]dehydroepiandrosterone[³⁵S]sulphate into brain tissue elements from the circulatory system and its metabolic fate in the brain were studied in developing rats. Approximately 0.037 % of [³H] and 0.023% of [³⁵S] were incorporated into the brain within 15 min after the intracardiac injection of the labelled steroid. More than one-half of the incorporated [³H] was recovered as free steroid, whereas the rest was recovered as sulphate. The ³H/³⁵S ratio in the sulphate fraction suggested that the sulphate entered the brain with the sulphate linkage intact. Upon intracerebral injection of the double-labelled steroid, approximately 6 per cent of the radioactivity was recovered in the brain at 30 min after the injection and 1 per cent was recovered at 1 h after the injection. Of the remaining radioactivity recovered from the brain, 5 per cent was found in the free steroid fraction, probably formed by hydrolysis of the sulphate; 90 per cent was in the sulphate ester fraction; and the rest was in the fraction of more polar compounds. To identify the metabolites, [4-¹⁴C]dehydroepiandrosterone sulphate was injected into the rat brain. Significant amounts of radioactivity were found in androstenediol sulphate, which was isolated from the brain. This compound was apparently derived from dehydroepiandrosterone sulphate by reduction of the 17-keto group to a 17β-hydroxyl group without prior hydrolysis. There was suggestive evidence that free androstenediol was also formed in the brain in this experiment.     

Diffusion of intracerebrally injected [1-14C]arachidonic acid and [2-3H]Glycerol in the mouse brain

[2-³H]Glycerol and [1-¹⁴C]arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. [2-³H]Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of [1-¹⁴C]arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in [2-³H]glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Polyamine turnover in different regions of rat brain

The dynamics of the formation and disappearance of polyamines in rat brain have been examined after intraventricular administration of a tracer dose of [³H]putrescine. After 2 days [³H]putrescine was no longer detectable in any brain region examined. [³H] Spermidine and [³H] spermine were formed in all brain areas. In the midbrain, hypothalamus and cerebellum (regions which manifested the greatest initial accumulation of tritium) the specific radioactivity of spermidine declined with a half-life of 16-19 days. However, in areas with a low initial accumulation of tritium (the medulla-pons, internal capsule, cerebral cortex and corpus striatum) the specific radioactivity of spermidine changed very little between 2 and 19 days after the putrescine administration. Levels of [³H]spermine increased continuously in all brain areas for a 14-day period after the putrescine injection.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.     

The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [³H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [³H]guanosine and were killed 5–45 min later. [³H]tRNA was extracted, partially purified, and hydrolyzed to yield [³H]-guanine and [³H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [³H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [³H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [³H]1-methylguanine represented up to 35% of the total [³H]methyl guanine radioactivity in astrocyte [³H]tRNA, while it became only negligibly labeled in brain [³H]tRNA. Conversely, brain [³H]tRNA contained more [³H]N²-methylguanine than did astrocyte [³H]tRNA. Approximately equal proportions of [³H]7-methylguanine were found in the [³H]tRNAs of both neural systems. The [³H]methylguanine composition of brain [³H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [³H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA^lys and tRNA^phe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.     

Axonal transport in nigro-neostriatal neurons during morphine tolerance development and abstinence in rats.

Axonal transport of [³H]protein in the nigro-neostriatal pathway in rats was examined during acute and chronic morphine administration and during morphine abstinence. Two days after a microinjection of [³H]lysine into the left substantia nigra zona compacta, more than 95% of the radioactivity present in the rat forebrain was protein-bound. Examination of frozen frontal brain sections revealed that 80–90% of the labelled protein of the injected side was located in brain areas traversed by the nigro-neostriatal pathway. As a positive control, intranigrally administered colchicine reduced the amount of [³H]protein transported after 5 days to the nucleus caudatus-putamen (neostriatum) to approx 18-26% of control. In animals rendered morphine-dependent by subcutaneous implantation of tablets containing 75 mg of morphine base, 27–86% more radioactivity accumulated in the neostriatum at 3, 4 and 5 days after [³H]lysine injection. In contrast, 23–48% less radioactivity was recovered in the neostriatal areas of animals withdrawing from morphine 24 h after [³H]lysine. Gel electrophoresis of soluble and particulate [³H]protein fractions from neostriatal tissues indicated that the gel patterns of radioactivity were not altered by chronic morphine administration. Neither morphine administration nor morphine abstinence altered the rate or amount of [³H]lysine incorporation into protein of the substantia nigra. These data demonstrate that chronic morphine administration was accompanied by a generalized increase in the amount of labelled protein transported to the neostriatum but the procedure was not sufficiently sensitive to detect a minor qualitative alteration of any particular protein(s). Furthermore, these data suggest that either the capacity or the rate of nigro-neostriatal protein transport may be increased during chronic morphine administration in the rat.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Glyceride metabolism in cultured cells dissociated from rat cerebral cortex

Abstract— [1-¹⁴C]stearic acid and [2-³H]glycerol were rapidly taken up and esterified into triacylglycerol and phospholipids by rat brain cells cultivated in monolayers. Expressed in terms of pool size, the incorporation of glycerol and stearate into triacylglycerol was 6- and 8-fold, respectively, higher than the incorporation into the choline phosphoglycerides. Tritium-labelled glycerol in both triacylglycerol and glycerophosphatides was diluted more rapidly than the [¹⁴C] labelled fatty acids. Chase experiments indicated a transfer of fatty acid from one lipid class to another, mainly from triacylglycerol to phospholipids, with no apparent loss of radioactivity. The accumulation of triacylglycerol in the brain cells was a function of both the presence of exogenous fatty acids in the culture medium and the metabolic needs of the cells; as long as the cells were involved in active formation of membranes the proportion of triacylglycerol was relatively small; its concentration increased while cell division slowed down in older, fully monolayered cultures.     

CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.     

Accumulation of the labdane diterpene Marrubiin in glandular trichome cells along the ontogeny of Marrubium vulgare plants

The content of the diterpene Marrubiin was assessed by GC-FID in leaves of Marrubium vulgare plants along their ontogeny. Maximum accumulation occurred just before flowering time and in fully expanded leaves. After feeding the plants with radio labeled [³H]-geranyl geranyl diphosphate, up to 70% of the radioactivity was recovered in HPLC-Rt coincidental with authentic Marrubiin, which was also characterized by GC-EIMS, thus confirming that the biosynthesis of Marrubiin proceeds through the 1-deoxy-D-xylulose-5-phosphate pathway. The major accumulation of radioactivity occurred in glandular trichome cells, and the product remained stable throughout.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

METHYLHISTAMINE: EVIDENCE FOR SELECTIVE DEAMINATION BY MAO B IN THE RAT BRAIN IN VIVO

Abstract— A new method has been developed for the separation of histamine and its metabolites after intracisternal injection of [³H]histamine into the rat brain, involving solvent extraction and subsequent thin-layer chromatography. The effect of graded doses of the MAO inhibitors deprenil and pargyline, which at relatively low doses inhibit preferentially the B form (phenethylamine deaminating) of the enzyme, and clorgyline, which mainly inhibits the A form (serotonin, noradrenaline and dopamine deaminating) on the brain levels of intracisternally injected [³H]histamine and its labelled metabolites was studied and compared to MAO A and B activity as determined with the substrates serotonin and phenethylamine, respectively. In addition, the time-course of the effects of a single dose of pargyline (50mg/kg subcutaneously) was investigated. No [³H]imidazoleacetic acid could be detected in any of the control or treated animals. [³H]Histamine accounted for 9–12% of the total extracted radioactivity and this was not altered significantly by pretreatment with any of the MAO inhibitors up to high doses, at which both MAO A and B activities were completely inhibited. In the controls, 40–43% of the total extracted radioactivity was [³H]methylhistamine and 28–30% was [³H]methylimidazoleacetic acid. Deprenil and pargyline caused [³H]methylhistamine levels to increase in a dose-dependent manner up to about 150% of control levels and those of [³H]methylimida-zoleacetic acid to decrease concomitantly to about 10% of control levels. Clorgyline in doses up to 10 mg/kg subcutaneously (s.c.) had no effect on the levels of these two metabolites. The dose-response curves of the effects of deprenil and pargyline on [³H]methylimidazoleacetic acid levels were congruent with those of the MAOI effects on MAO B activity and not with those on MAO A activity. Pargyline (50 mg/kg s.c.) had a long lasting effect on the accumulation of [³H]methylhistamine and [³H]methylimidazoleacetic acid. Recovery occurred within 21 days, and the half-lives observed were 5.3 and 5.6 days, respectively. This compares well to the half-life for the recovery of MAO B activity reported earlier after the same dose of pargyline (5.5 days). These results suggest that methylhistamine is metabolized selectively by MAO B in rat brain. Moreover, the fact that clorgyline, at doses where phenethylamine deamination is already considerably inhibited, did not affect the deamination of methylhistamine, suggests that the latter is an even more selective substrate for MAO B than phenethylamine itself. Therefore, small doses of deprenil (0.3–3 mg/kg s.c.) or pargyline (1–3 mg/kg) can be used to influence histamine catabolism without interfering with catecholamine or serotonin deamination.     

Differential Utilization of the Ethanolamine Moiety of Phosphatidylethanolamine Derived from Serine and Ethanolamine during NGF-Induced Neuritogenesis of PC12 Cells

Neurite elongation involves the expansion of the plasma membrane and phospholipid synthesis. We investigated membrane phosphatidylethanolamine (PE) biosynthesis in PC12 cells during neurite outgrowth induced by nerve growth factor (NGF). When PE was prelabeled with [³H]ethanolamine and the radioactivity was chased by incubation with 1 mM unlabeled ethanolamine, the radioactivity of [³H]PE steadily declined and [³H]ethanolamine was released into the medium in NGF-treated cells during neurite outgrowth; in the absence of unlabeled ethanolamine, the radioactivity of [³H]PE remained relatively constant for at least 24 hr. In undifferentiated cells but not in NGF-treated cells, [³H]phosphoethanolamine accumulated in significant amounts during pulse labeling, and was converted partly to PE but largely released into the medium irrespective of incubation with unlabeled ethanolamine. The decline in the radioactivity of [³H]PE and release of [³H]ethanolamine following incubation with unlabeled ethanolamine were also observed in undifferentiated cells. Thus, the ethanolamine moiety of PE derived from ethanolamine is actively recycled in both differentiated and undifferentiated cells. When PE was derived from [³H]serine through phosphatidylserine (PS) decarboxylation, the decrease in radioactivity of [³H]PE and release of [³H]ethanolamine into the medium following incubation with unlabeled ethanolamine were observed only in NGF-treated cells, but not in undifferentiated cells, indicating that the ethanolamine moiety of PE derived from PS is actively recycled only in the cells undergoing NGF-induced neuritogenesis. Thus, in PC12 cells, the ethanolamine moiety of PE derived from PS is regulated differently from that of PE derived from ethanolamine.     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography