MASS FRAGMENTOGRAPHIC DETERMINATION OF SOME ACIDIC AND ALCOHOLIC METABOLITES OF BIOGENIC AMINES IN THE RAT BRAIN

—A mass fragmentographic procedure is described for the simultaneous quantification of a number of deaminated metabolites derived from tyramine, octopamine, dopamine, and norepinephrine. With this method, several of the metabolites were measured in normal rat brain. The results support the central nervous system origin of tyramine, octopamine and their metabolites. The concentration of the dopamine metabolite, homovanillic acid, in the rat brain was found to be about 15% higher than that of dihydroxyphenylacetic acid. As for the metabolites of norepinephrine, vanilmandelic acid concentration was found to be about 5% that of 3-methoxy-4-hydroxyphenylglycol. The possible role of vanilmandelic acid in the CNS metabolism of norephrine is discussed.     

DISTRIBUTION OF BIOGENIC AMINES AND RELATED ENZYMES IN THE RAT PITUITARY GLAND

Abstract— Biogenic amines and related enzymes were quantitatively measured in the pituitary gland of the rat. The sensitivity of the assays used allows the determinations to be performed in single pituitary lobes. Relatively high values of histamine and serotonin were found in all three lobes, with higher amounts in the posterior and intermediate lobes. Highest catecholamine concentrations were detected in the posterior lobe, and only very low amounts of dopamine were measured in the anterior lobe. Throughout the gland, norepinephrine concentrations were low, about one-tenth that of dopamine. Tryptamine could not be detected. High levels of A and B monoamine oxidase were found in all three pituitary lobes. Tyrosine hydroxylase activity was measured in the posterior and intermediate lobes, but was not detected in the anterior lobe. Tryptophan hydroxylase was present in all three pituitary lobes. A relatively low catechol- O -methyltransferase activity was found in the anterior lobe, and none was detected in the intermediate and posterior lobe. Choline acetyltransferase, dopamine-β-hydroxylase and phenylethanolamine- N -methyltransferase activities could not be detected.     

THE SITE OF ORIGIN OF ELECTRICAL RESPONSES IN VISUAL CELLS OF THE LEECH, HIRUDO MEDICINALIS

In leech visual cells the presumed light-absorbing structures are microvilli arising from the membrane of what would seem to be a large intracellular vacuole. This vacuole, however, is an extracellular compartment, since it communicates with the intercellular spaces through narrow channels. Therefore, the membrane of the microvilli is—as in other invertebrate visual cells—a part of the cell membrane. Visual responses recorded with an electrode within the vacuole were compared with the intracellular recordings. Following illumination the vacuole becomes negative with respect to the outside fluid, while the cells are depolarized. This finding indicates that inward current penetrates the cell through the microvillar membrane. It is concluded, therefore, that the electrical response (receptor potential) originates as a result of changes in the properties of the light-absorbing membrane.     

BRAIN CONCENTRATIONS OF BIOGENIC AMINES AND THEIR METABOLITES IN TWO TYPES OF PYROGEN-INDUCED FEVER IN RABBITS

The effects of two bacterial pyrogens (gonococcus vaccine and E. coli lipopolysaccharide) on brain and CSF concentrations of the biogenic amines and their major metabolites were compared in rabbits. Both pyrogens induced a similar fever, but only the lipopolysaccharide elicited marked biochemical alterations. The main changes observed were: a decrease in the concentrations of acid metabolites of dopamine during the upward course of the first peak of fever; a decrease in the hypothalamic concentrations of noradrenaline at the second peak of fever; a general increase in the metabolites of biogenic amines around the second peak and during the fall of fever. The results led to the conclusion that no significant correlation exists between body temperature and brain amine levels in rabbits. The alterations observed could be a consequence of the fever itself when considered in terms of trauma to the organism.     

THE EFFECTS OF OUABAIN AND THE ACTIVATION OF NEURAL MEMBRANE ATPase BY BIOGENIC AMINES

Abstract— The effects of 10⁻⁵ m -noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) on the activities of Na⁺-K⁺ ATPase (EC 3.6.1.3) were studied in synaptic membranes from 6 regions of the rabbit brain. NA and 5-HT stimulated the synaptic membrane Na⁺-K⁺ ATPase from the cerebrum, but none of the amines influenced the activity of this enzyme in the other brain regions. The Na⁺-K⁺ ATPase activity of the cerebral synaptic membrane isolated at the 0.8/0.9 m & 0.9/1.0 m interphase of a sucrose density gradient was increased two-fold by 10⁻⁵ m -NA and 5-HT. The Na⁺-K ⁺ ATPase recovered at the 1.0/1.2 m interphase was not influenced by NA, DA or 5-HT. NA, DA and 5-HT did not activate the Mg ATPase of synaptic membranes from any of the 6 brain regions or whole brain synaptic vesicles. The cortex synaptic membrane (Na⁺-K⁺) ATPase is postulated to have a direct role in the uptake of the biogenic amines. An indirect role is proposed for this enzyme in amine uptake into brain stem.     

两种常见海水鱼高温贮存过程中挥发性盐基氮和生物胺含量变化

棘头梅童鱼(Collichthys lucidus)和龙头鱼(Harpodon nehereus)是我国沿海常见的两种小型海水鱼, 常被作为水产动物饵料, 也可被人类食用。研究检测了这两种鱼在30℃下贮存48h 每隔6h 的挥发性盐基氮(T-VBN)和9 种生物胺(尸胺、腐胺、组胺、酪胺、5-羟色胺、亚精胺、精胺、多巴胺、章鱼胺)的含量变化, 并对这两种鱼的T-VBN 和生物胺含量与时间的相关性进行分析, 为水产品类饵料安全投喂和人类食品安全提供基础资料。结果表明: 两种鱼在相同贮存条件中T-VBN 和生物胺含量均存在一定差异。T-VBN 含量随着贮存时间的延长而逐渐增加, 棘头梅童鱼T-VBN 含量从0h 的8.19 mg/100 g 增加到48h 的568.05 mg/100 g,龙头鱼从0h 的13.16 mg/100 g 增加到48h 的361.34 mg/100 g, 棘头梅童鱼增长值显著高于龙头鱼(PPPP>0.05);多巴胺在两种鱼体内均未检测到。这两种鱼体内T-VBN、腐胺、尸胺、组胺、酪胺含量与时间的相关性均极其显著(P<0.01)。       

大鼠胚胎和成年组织中EGFR基因转录分析

本文报告用2.4Kb EGFR cDNA PE7为探针,分析大鼠胚胎发育过程中全胚组织、成年大鼠七种组织和再生肝组织中EGFR基因转录产物的水平。实验结果说明:(1)用人EGFRcDNA探针可以检测到大鼠肝脏细胞转录约为5.6Kb的EGFRmRNA。(2)大鼠胚胎发育过程中,EGFR基因转录活性显示骤然变化。10—12天胚胎中EGFR mRNA出现高峰。(3)成年大鼠肝、肾、肺、脑、脾、心脏和睾丸组织中均有EGFR基因的转录,转录水平各有差异,以肾为最高。(4)大鼠肝脏再生过程中,EGFR基因转录呈现时相调节的特征,部分肝切除刺激EGFR基因转录活性的增高,8小时达到高峰后又回落到接近正常的水平。这些EGFR基因转录的变化可能与大鼠组织和细胞的生长及分化的调控有关。     

BIOGENIC AMINES AND THEIR METABOLITES AS SUBSTRATES FOR PHENOL SULPHOTRANSFERASE (EC 2.8.2.1) OF BRAIN AND LIVER

Phenol sulphotransferase activity in homogenates of rat liver and brain was determined spectrophotometrically. Rat liver had about 100-fold more phenol sulphotransferase activity than brain; however, both tissues showed about the same spectrum of activity towards the phenolic compounds tested. Dopamine and its acidic and neutral metabolites and the neutral metabolites of norepinephrine were the compounds most readily sulphury-lated in vitro. They were also the compounds most readily sulphurylated in vivo when they were injected intraventricularly together with labelled Na₂SO₄. When labelled Na₂SO₄ was injected alone, we detected conjugation of endogenous phenols. One of the compounds formed was identified by its chromatographic characteristics as 3-methoxy-4-hydroxy-phenylethyleneglycol sulphate. We detected other conjugates which appeared to be the sulphate esters of 3,4-dihydroxyphenylethyleneglycol; 3,4-dihydroxyphenylacetic acid; and homovanillic acid. In brain, sulphate conjugation may be a major route of metabolism for many of the phenolic compounds related to the biogenic amines and possibly for phenolic drugs which enter the brain.     

MASS FRAGMENTOGRAPHY OF PHENYLETHYLAMINE, m- AND p-TYRAMINE AND RELATED AMINES IN PLASMA, CEREBROSPINAL FLUID, URINE, AND BRAIN

—A mass fragmentographic method for the assay of phenylethylamine (PEA) and a number of related amines in several biological materials is described. The gas chromatographic column employed for this analysis is a 12ft 1/8 in. o.d. steel column packed with 0.5% OV₂₂+ 2% SE54 + 1% OV₂₁₀ coated on 80/100 mesh chromosorb W (HP). The mass spectral characteristics of these amines are illustrated, compared, and discussed. Of the various monoamines which could be measured, only PEA, m- and p-tyramine were detected in measurable quantities. Phenylethanolamine and p-octopamine were found in trace amounts in urine, plasma, cerebrosponal fluid, and rat brain. No diurnal variation in the urinary excretion of PEA, m- and p-tyramine was observed. Plasma concentration of PEA or p-tyramine did not significantly change 1 h after eating a breakfast. Furthermore, consuming 200 g of Cadbury milk chocolate containing about 1 mg of PEA, 0.1 mg of phenylethanolamine and 10 mg of p-tyramine did not significantly alter urine excretions of these three amines. In the brain, as has been reported by others, we found that PEA and p-tyramine are not evenly distributed and that the highest concentrations are found in the hypothalamus and caudate. From the results obtained we concluded that PEA, m- and p-tyramine are probably produced from endogenous sources and that the direct contribution of diet to their urine excretion is small.     

NET PRODUCTION OF γ-AMINOBUTYRIC ACID IN ASTROCYTES IN PRIMARY CULTURES DETERMINED BY A SENSITIVE MASS SPECTROMETRIC METHOD

A sensitive and specific high resolution mass spectrometric method, using a deuterium labelled internal standard, was developed for the quantitation of GABA in biological materials. GABA was converted to the DNS-γ-butyrolactam, isolated by thin layer chromatography and quantitated by the integrated ion current procedure. The method was used to measure the GABA content of cultured astrocytes (0.0545 nmol/mg protein, depending upon the culturing conditions), of fresh media (0.02–0.04 μM) and of conditioned media (after culturing in the absence of added GABA: 0.02–1.8 μM, depending upon the culturing conditions). An estimate of the rate of GABA production was obtained from the concentration in conditioned media and in the cells when GABA degradation was inhibited by AOAA (13 μM). The production of GABA was negligible in the cells grown under ordinary conditions but increased to 0.3 nmol/h per mg protein in cells which had been grown in the presence of 0.25 mM- dibutyryl cyclic AMP for 1 week. This value is of the same order of magnitude as the GAD activity observed in extraneurónal tissue. However, the GABA production measured mass spectrometrically was much lower than that of labelled GABA from [U-¹⁴C]glutamate. The latter value was non-reproducible and varied from one batch of radioactive glutamate to another.     

中国树鼩下丘脑视上核和室旁核内加压素能和催产素能神经元的形态计量学分析

采用免疫组织化学ＰＡＰ／ＤＡＮ－镍加强技术和形态计量法研究了中国树鼩下丘脑加压素（ＶＰ）能和催产素（ＯＴ）能视上核主部（ＳＯＮｐ）、视上核交叉后部（ＳＯＮｒ）和室旁核（ＰＶＮ）的平均矢轴长度,ＳＯＮｐ,ＳＯＮｒ和ＰＶＮ内ＶＰ能和ＯＴ能神经元胞体截面积的平均值;ＳＯＮｐ,ＳＯＮｒ和ＰＶＮ的ＶＰ能和ＯＴ能神经元总数;各校团的各个系列节段内ＶＰ能和ＯＴ能神经元的比较计数。发现,ＳＯＮｐ前１／３部分ＯＴ能神经元明显多于ＶＰ能的,而后２／３部分则相反;ＳＯＮｒ前２／３部分ＶＰ能神经元明显多于ＯＴ能的,而后１／３部分两者差别不明显;ＰＶＮ前２／３部分ＯＴ能神经元明显多于ＶＰ能的,而后１／３部分则相反。本文就中国树下丘脑ＳＯＮ和ＰＶＮ内ＶＰ能和ＯＴ能神经元的形态计量学资料与大鼠的进行了比较。     

DISTRIBUTION AND TURNOVER OF OCTOPAMINE IN TISSUES

Abstract— Octopamine is a normally occurring amine in several species of animals. Particularly high concentrations are found in the crustacean central nerve cord. In the rat it is specifically localized to sympathetic nerve endings, has a subcellular distribution similar to that of norepinephrine, and is asymmetrically distributed in the CNS. It has a turnover rate in heart about six times that of norepinephrine. The physiological role of octopamine has not been established but it appears likely that it is a cotransmitter together with norepinephrine in the peripheral sympathetic nervous system.     

PHYLOGENETIC ANALYSIS OF LEARNING-RELATED NEUROMODULATION IN MOLLUSCAN MECHANOSENSORY NEURONS

In spite of significant advances in our understanding of mechanisms of learning and memory in a variety of organisms, little is known about how such mechanisms evolve. Even mechanisms of simple forms of learning, such as habituation and sensitization, have not been studied phylogenetically. Here we begin an evolutionary analysis of learning-related neuromodulation in species related to the well-studied opisthobranch gastropod, Aplysia californica. In Aplysia, increased spike duration and excitability in mechanosensory neurons contribute to several forms of learning-related changes to defensive withdrawal reflexes. The modulatory transmitter serotonin (5-hydroxytryptamine, or 5-HT), is thought to play a critical role in producing these firing property changes. In the present study, we tested mechanosensory homologs of the tail-withdrawal reflex in species related to Aplysia for 5-HT-mediated increases in spike duration and excitability. Criteria used to identify homologous tail-sensory neurons included position, relative size, resting electrical properties, expression of a sensory neuron-specific protein, neuroanatomy, and receptive field. The four ingroup species studied (Aplysia californica, Dolabella auricularia, Bursatella leachii, and Dolabrifera dolabrifera) belong to two clades (two species each) within the family Aplysiidae. In the first clade (Aplysia/Dolabella), we found that the tail-sensory neurons of A. californica and tail-sensory homologs of a closely related species, D. auricularia, responded to bath-applied serotonin in essentially similar fashion: significant increases in spike duration as well as excitability. In the other clade (Dolabrifera/Bursatella), more distantly related to Aplysia, one species (B. leachii) showed spike broadening and increased excitability. However, the other species (D. dolabrifera) showed neither spike broadening nor increased excitability. The firing properties of tail-sensory homologs of D. dolabrifera were insensitive to 5-HT over a wide range of concentrations. We also performed experiments on two outgroup species (Akera bullata and Bulla gouldiana) and found that spike duration was unaffected by 5-HT, whereas excitability was increased. This study suggests that 5-HT-induced spike broadening arose more recently in opisthobranch evolution, whereas 5-HT-induced excitability increase is a more ancestral trait that may have been expressed in the earliest opisthobranchs. Both traits are absent in the aplysiid species D. dolabrifera, demonstrating that a lineage can lose learning-related mechanisms. The phylogenetic variation observed in the present study presents the opportunity to test general models about learning mechanisms and their evolution in unique ways.     

A SENSITIVE ENZYMATIC-ISOTOPIC METHOD FOR THE ANALYSIS OF TYRAMINE IN BRAIN AND OTHER TISSUES

Abstract— An enzymatic-isotopic assay for the measurement of tyramine with a sensitivity of 1.0 ng has been developed. Using this assay, the endogenous content of tyramine in various tissues from adult rats has been determined. The highest tyramine content was found in rat heart atria, followed by salivary gland, kidney, and brain. Within the brain the distribution of tyramine is heterogeneous and the highest tyramine content was localized in the striatum.