BIOCHEMICAL CHARACTERIZATION OF β-N-OXALYL-l-α,β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN AS AN EXCITANT AMINO ACID

Abstract— β- N -Oxalyl- l -α,β-diaminopropionic acid (ODAP), the toxin isolated from the seeds of Luthyrus sativus produces head retraction, tremors and convulsions when injected into a variety of experimental animals. In 12-day-old rats, it has been found that the convulsive behaviour is accompanied by profound biochemical changes in the brain. The brain homogenates prepared from ODAP injected animals show a higher rate of respiration. There is a decrease in the brain glucose, glycogen, ATP, phosphocreatine and acetylcholine levels of the convulsing animals. The inorganic phosphate, lactic acid and acetylcholineesterase levels increase. These results establish that ODAP is a typical convulsant.     

STUDIES ON THE TISSUE AND SUBCELLULAR DISTRIBUTION OF β-N-OXALYL-l-α, β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

Abstract— β- N -Oxalyl- l -α, β-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin can be detected in significant concentrations in the synaptosomal fractions isolated from young rat brain and adult monkey spinal cord, when these animals manifest neurological symptoms after ODAP administration. However, isolated synaptosomes fail to exhibit any transport system for ODAP uptake. ODAP administered in vivo appears to get localized in a population of synaptosomes which exhibit a high affinity uptake system for glutamate.     

Mechanism of Action of β-N-Oxalyl-L-α,β-diaminopropionic Acid, <Emphasis Type="Italic">Lathyrus sativus</Emphasis> Neurotoxin: Effect on Brain Lysosomes

β-N-Oxalyl-L-α,β-diaminopropionic acid (ODAP), the toxic amino-acid isolated from the seeds of Lathyrus sativus^1,2, has been implicated in human neurolathyrism, a crippling disease found in central India and attributed to the consumption of this vetch³. ODAP causes convulsions in young animals when injected intraperitoneally^4,5. The toxin induces hind leg paralysis in monkeys when introduced into the immediate environment of the brain through the lumbar route⁶. ODAP is a potent excitant amino-acid in the Betz cells of cat spinal cord⁷ and produces biochemical changes in the rat brain typical of an excitant amino-acid⁸. Chronic ammonia toxicity has been implicated in the mode of action of ODAP, leading to convulsions in young rats⁹. Protein degradation in the brain may be an early event in ODAP-induced convulsions¹⁰. Nucleotides have also been found to accumulate in the brain of young rats injected with ODAP (our unpublished data). Thus, a generalized increase in the catabolism of protein and nucleic acids in ODAP induced convulsions has been indicated by earlier investigations. We have examined the effect of ODAP on chick brain lysosomes possibly leading to a liberation of acid hydrolases.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

AGE-DEPENDENT VARIATIONS OF THE HUMAN N-ACETYL-β-D-HEXOSAMINIDASES

The total N-acetyl-β-d -hexosaminidase (hexosaminidase A plus B) activity of the human brain increases during life until the age of 70 years to about twice the activity found in the foetal brain. This activity roughly parallels the increasing level of brain ganglio-sides. Simultaneously, the ratio between the hexosaminidases A and B changes from approx. 3.4 to 1.1 as established by the microscale isoelectric focussing of the enzymes from crude extracts. Age-dependent variations can be also demonstrated for other glycosidase activities: The β-d -glucosidase is shown to increase, the α-d -mannosidase and α-l -fucosidase to decrease during life. The enzyme values are related to the protein content, the age-dependent shift of which we have determined in the human brain. In different organs and body liquids distinct differences are found for the levels of the total hexosaminidase activity as well as for the hexosaminidase pattern. The relation of the hexosaminidase activity to the ganglioside degradation during the human development is discussed.     

THE EFFECT OF β,β''-IMINODIPROPIONITRILE AND ANAESTHESIA ON SOME ADENINE NUCLEOTIDES OF RAT BRAIN AND RETINA

—A significant increase in the retinal ATP content of anaesthetised rats was found 6 days after administration of β, β′-iminodipropionitrile (IDPN). With the development of retinal dystrophy variable ATP levels were observed from the 8th to the 12th day and low values were recorded on the 17th and 21st days. At 8 days after IDPN administration the ATP content of anaesthetised rat brain was significantly increased with slight decreases in ADP and AMP levels. The differences in the level of these adenine nucleotides in unanaesthetised and anaesthetised rat brain were not significant before or after IDPN administration. These results were related to previous experiments on the action of IDPN on the electroretinal response and the later development of a retinal microangiopathy. It was suggested that IDPN has a primary neurotoxic effect followed by the development of vascular morphological changes.     

ENZYMATIC ISOTOPIC ASSAY FOR AND PRESENCE OF β-PHENYLETHYLAMINE IN BRAIN

Abstract— An enzymatic isotopic assay for the measurement of β-phenylethylamine in brain, with a sensitivity of 100-200 pg, has been developed. With this assay, the endogenous β-phenylethylamine content (1.5 ng/g) in the rat brain has been determined. Phenylalanine administration increases the brain levels of this amine; inhibition of monoamine oxidase causes a 40-fold increase in brain β-phenylethylamine. After a combined treatment with a monoamine oxidase inhibitor and phenylalanine, the β-phenylethylamine content in the brain increases to about 400-fold. This increase can be blocked by the central decarboxylase inhibitor NSD-1055. p-Chlorophenylalanine also increases β-phenylethylamipe concentration in the brain, and this effect is potentiated by a simultaneous administration of phenylalanine.     

BIOCHEMICAL CHANGES IN β,β''-IMINODIPROPIONITRILE-TREATED RAT BRAIN AND PREVENTION OF TOXICITY BY ETHIONINE

The administration of β,β′-iminodipropionitrile (IDPN) to rats, either in five daily injections of 30 mg, or in a single injection of 100 mg/100 g body wt., resulted in the development of severe damage to the central nervous system and retinal vasculature. These changes were prevented by the daily intraperitoneal injection of 24 mg dl -ethionine/100 g body wt. Significant increases in the oxygen uptake of IDPN-treated rat brain were found when measured in the presence of succinate or glutamate as substrates. IDPN (5 mm ) did not affect the oxygen uptake of brain homogenates in vitro when measured in the presence of the same substrates. The cytochrome oxidase activity of rat brain was not significantly changed by in vivo administration of IDPN, nor by the presence of 5 mm -IDPN in vitro. The lactate content of the IDPN-treated rat brain was significantly increased by the eighth day. There were no changes in the dry wt., total protein, lipid or phospholipid content of the IDPN-treated rat brain, even after 4 weeks. These findings are discussed with reference to previous experiments on the toxic action of IDPN on the central nervous system and retinal vasculature.     

β-NEUROTOXIN REDUCES NEUROTRANSMITTER STORAGE IN BRAIN SYNAPSES

β-neurotoxin, a component of Bungarus multicinctus venom, is known to cause neuromuscular blockade by first increasing the rate of spontaneous ACh release and then inhibiting the nerve impulse-induced release of ACh. We report that the toxin also affects the storage of several neurotransmitters in rat brain and it is active on synaptosomes and brain minces. Synaptosomes prepared from brain tissue that had been treated with β-toxin have a reduced ability to accumulate radioactive NE, GABA, serotonin and the ACh precursor, choline. The toxin also causes a release of previously accumulated NE and GABA from synaptosomes, suggesting that the storage process rather than the uptake transport process is affected. The toxin does not contain phospholipase A, phospholipase C, protease or hyaluronidase activity.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

EFFECT OF THYROID DEFICIENCY ON THE LEVELS OF SEVERAL ENZYMES IN RAT BRAIN

—Activities of acid phosphatase, alkaline phosphatase and β-glucuronidase have been estimated in the brain tissues, using various subcellular particles, in growing thyroidectomized rats and also using cytoplasmic extracts free from debris and nuclear fraction in young hypothyroid animals. Hepatic glucose-6-phosphate dehydrogenase activity was markedly reduced after thyroidectomy but the enzyme was brought back to normal levels by thyroxine treatment. There was no change, however, in the activity of neural glucose-6-phosphate dehydrogenase after thyroidectomy. In the thyroidectomized animals an increase only in the free acid phosphatase activity in the neural synaptosomes was found and this increase in activity was not counteracted by administration of thyroxine. In the hypothyroid young animal β-glucuronidase, acid phosphatase and alkaline phosphatase activities were found to be affected during development.     

ENTRY OF β-OXALYL-l-α,β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN INTO THE CENTRAL NERVOUS SYSTEM OF THE ADULT RAT, CHICK AND THE RHESUS MONKEY

Abstract— A comparative study of the tissue disposition of the L. sativus neurotoxin has been done using [³H]0DAP with special reference to the CNS in normal and Diamox treated (acidotic) adult rhesus monkeys and also in the day old chick, a species susceptible to the neurotoxin, and the adult rat which is refractory to it. The neurotoxin was detected in the CNS of the acidotic monkey and also in the normal monkey in nearly the same quantity. The neurotoxin was largely localised in the lumbosacral region of the spinal cord. The amount of radioactive ODAP in the CNS following its intraperitoneal injection was twice that seen following its intravenous injection.
The concentrations of the neurotoxin 90 min after injection in the CNS of the adult rat and the day old chick were almost the same and increased 2 fold by 24 h. No radioactive metabolite of ODAP could be detected in either the tissues or the urine of the different species. Species (and age) differences in susceptibility to the L. sativus neurotoxin is thus independent of its entry into the CNS.     

Hydrolysis of Galactose from Glycolipids and Glycoprotein by Young Rat Brain β-Galactosidases Immobilized to Sepharose 4B

An extract of glycosidic enzymes from young rat brain was immobilized to cyanogen bromide-activated Sepharose 4B. Most glycosidases retained approximately 10-25% of their activities after immobilization. Immobilized β-galactosidases were used repeatedly without detectable loss of enzyme activity in the hydrolysis of p-nitrophenyl-β-d -galactopyranoside. In addition to the synthetic substrate, the immobilized rat brain β-galactosidases could also hydrolyze galactose from lactose, galactosylcerebroside, asialofetuin, and GM₁-ganglioside. The hydrolysis of GM₁- to GM₂-ganglioside was confirmed on TLC.     

High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β-Lyase) and ω-Amidase in the Choroid Plexus of Rat Brain

Abstract: Certain halogenated hydrocarbons, e.g., dichlo-roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine-S-conjugate β-lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine-S-conjugate β-lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω-amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α-keto acid substrates. The ω-amidase also has a broad specificity; presumably, however, the natural substrates are α-ketoglutaramate and α-ketosuccinamate, the α-keto acid analogues of glutamine and aspara-gine, respectively. The high activities of both glutamine transaminase K and ω-amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l -glutamine and α-keto acid α-ketoglutarate and l -amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.     

β‐1,3‐Glucanases in wheat and resistance to the Russian wheat aphid

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) is one of the most destructive insect pests of cereals world-wide. Although resistant cultivars have been bred, the biochemical mechanism of resistance is unknown. The aim of this work was to gain information on the mechanism of resistance which could contribute to more directed breeding of resistant cultivars in the future. The effect of RWA infestation on the inter- and intracellular β-1,3-glucanase activities was studied in different resistant wheat (Triticum aestivum L.) cultivars containing the Dn-1 gene for RWA resistance and corresponding near-isogenic susceptible cultivars. The activity was determined spectrophotometrically by measuring the release of glucose from laminarin. Infestation differentially induced the intra- and intercellular activities to much higher levels in resistant than susceptible cultivars within 48 h. According to immunological studies induced enzyme activities were due to increased protein levels. The intracellular β-1,3-glucanase contained about 8% exo-activity. The exo-activity made an insignificant contribution to the intercellular activity. The genetic background into which the resistance gene was bred did affect the level of activity that corresponded to the resistance performance. Seven apoplastic isoforms of β-1,3-glucanase, varying from acidic to basic, were resolved by isoelectric focusing. All isoenzymes were equally induced and no specific one could be linked to resistance or susceptibility. The RWA induced β-1,3-glucanase activity in resistant cultivars closely resembles defence responses during pathogenesis and seems to be part of a general defence response like the hypersensitive reaction (HR), which confers resistance to the RWA. This knowledge might be helpful in future to identify genes for RWA resistance. The increased β-1,3-glucanase activity after RWA infestation might serve as an additional measure to biochemically trace resistance in crosses during breeding.     

Mode of Action of α, β Unsaturated Carbonyl Compounds

The toxicity of the α, β unsaturated carbonyl compounds (α, β UCC_s) (patulin, penicillic acid, parasorbic acid, tulipalin and plumbagin) towards Pythium sp. group F (Van Der Plaat -Niterinks 1981) was neutralized by the addition of an excess of cysteine. This suggests that the mode of action of these compounds could be due to a binding of the α, βi UCC_s to sulphydryl groups in enzymes or other macromolecules. Alcohol dehydrogenase (ADH), an enzyme with a sulphydryl group at the active site, was assayed spectrophotometrically and all the α, β UCC_s inhibited ADH.     

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.     

Antibodies Specific for α-N-Acetyl-β-Endorphins: Radioimmunoassays and Detection of Acetylated β-Endorphins in Pituitary Extracts

Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys⁹]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.     

THE SUBCELLULAR DISTRIBUTION OF β-PHENYLETHYLAMINE, p-TYRAMINE AND TRYPTAMINE IN RAT BRAIN

—The quantitative subcellular distribution of β-phenylethylamine, p-tyramine and tryptamine in rat brain was investigated using the mass spectrometric integrated ion current technique. More of the total cellular tryptamine was found to be associated with paniculate fractions than was the case for phenyiethylamine and p-tyramine but a significant amount of this tryptamine was found to be labile. Analysis of the particulate fractions indicated that each of the amines was localized predominantly in the crude P₂ pellet and that the bulk of this was associated with the synaptosomal (P₂B) fraction. Inhibition of monoamine oxidase systems with pargyline caused an increase in the level of all three amines in all fractions, but the increase was greater in the supernatant than in the combined particulate fractions. This treatment produced changes in the distribution of β-phenylethylamine and p-tyramine between the various particulate subcellular fractions but did not markedly alter the distribution of tryptamine between the same fractions.