CHARACTERIZATION OF HYPOTHALAMIC SUBCELLULAR PARTICLES CONTAINING LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPIN RELEASING HORMONE

Abstract— The 900 g supernatant fluid prepared from male rat hypothalamic homogenates was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone and luteinizing hormone releasing hormone in the gradient fractions were quantified by radioimmunoassays. TRH was associated with two populations of particles separable by means of nonequilibrium density centrifugation (100,000 g for 30min). However, after'equilibrium'centrifugation (100,000 × g for 180 min), a single peak of TRH was observed at 1.07 M-sucrose. Hypo-osmotic shock as well as treatment with 0.1% Triton X-100 or 0.1% deoxycholate (DOC) released TRH from both sets of particles. LRH, as TRH, was associated with two populations of particles which were separable by means of nonequilibrium density gradient centrifugation. After'equilibrium'centrifugation, both sets of LRH-containing particles banded at 1.27M-sucrose as a single symmetrical peak. Although 0.1% Triton X-100 released LRH from both populations of particles, hypo-osmotic shock or 0.1% DOC released LRH only from the large LRH-containing particles. The small LRH-containing particles were resistant to hypo-osmotic shock and to 0.1% DOC. Based on these criteria, it is concluded that in hypothalamic homogenates the TRH-containing particles and the large LRH-containing particles are synaptosomes. The small LRH-containing particles may be of different cellular and/or subcellular origin.     

促甲状腺激素释放激素(TRH)鞘内注射易化大鼠甩尾反射及其与吗啡的交互作用

本实验采用 Yaksh 和 Rudy 的慢性埋植套管鞘内注射的方法,观察了 TRH 对大鼠甩尾反射的直接作用以及与吗啡的相互作用。鞘内注射1μg、5μg和10μgTRH 能产生时程长的缩短甩尾潜伏期的效应,且这种效应呈剂量反应关系。在鞘内注射 TRH 的情况下,皮下或鞘内注射吗啡仍可产生吗啡的镇痛效应。先注射吗啡,TRH 不能翻转吗啡的延长甩尾潜伏期的效应。结合本文及文献上的其他结果说明:TRH 作用于脊髓靠近传出的部分,而吗啡则作用于脊髓靠近传入的部分。     

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.     

穹窿切断对大鼠下丘脑CRH表达的影响

目的观察穹窿切断对大鼠下丘脑促肾上腺皮质激素释放激素(CRH)表达的影响,探讨海马对HPA轴的抑制作用是否通过穹窿介导。方法成年健康雄性Wistar大鼠80只,随机分为穹窿切断组和假手术组,再分为0d、4d、7d、10d组,每组10只。建立大鼠穹窿切断模型,采用免疫组织化学和Westernblot技术检测穹窿切断组0d、4d、7d、10d时下丘脑CRH的表达变化和分布规律,并以假手术组相应时间段作为对照。结果穹窿切断组于7d时CRH表达升高,10d时升高显著。假手术组下丘脑仅有少量CRH表达。结论穹窿切断使CRH表达升高,HPA轴活动增强,海马对HPA轴的抑制作用减弱,揭示海马是通过穹窿纤维对HPA轴发挥抑制作用。     

丘脑下部促黄体素释放激素类似物(LHRH-A)对文昌鱼精子发生的影响:组织学及其超微结构的研究

本文报道将LHRH-A注射到文昌鱼体腔,可促使其精巢发育的实验结果。发育的精巢占实验动物总数的87％,没有变化者占17％。组织学和电镜观察指出LHRH-A可激发A型精原细胞分裂为B型精原细胞,促使初级精母细胞发育为次级精母细胞和精细咆。另外,LIRH-A有促使精于的释放的作用。 我们认为LHRH-A可激发文昌鱼的生精活动。     

阉割和睾丸酮替代对雄性老年大鼠下丘脑和血浆促黄体生成素释放激素水平的影响

应用放射免疫分析(RIA)技术比较了年青(2—3月龄)和老年(24—26月龄)雄性大鼠下丘脑和血浆促黄体生成素释放激素(LHRH)水平及其在睾丸切除(ORDX)和睾丸酮(T)替代下LHRH水平的变化。老年大鼠血浆T水平明显降低,下丘脑LHRH含量亦呈明显下降趋势,但血浆LHRH水平与年青大鼠十分接近。在ORDX和T替代下,两组动物血浆T水平没有明显差别,但老年大鼠下丘脑和血浆LHRH的变化率却不同程度地低于年青大鼠。上述结果提示,老年雄性大鼠下丘脑LHRH神经元系统的负反馈能力明显削弱,这也许是下丘脑-垂体-睾丸轴呈增龄性衰变的重要原因之一。     

大白鼠颌下腺促性腺激素释放激素受体(GnRHR)mRNA的RT-PCR和原位杂交的研究

本实验采用RT－PCR法探讨大白鼠颌下腺是否存在GnRH受体mRNA,并用原位杂交法对其细胞定位进行了研究。结果显示RT－PCR可扩增出大白鼠颌下腺GnRH受体mRNA的特异性片段,其碱基数与设计的一致,原位杂交发现颌下腺浆液性腺泡上皮细胞、颗粒曲管、排泄管及分泌管上皮细胞内有GnRH受体mRNA的杂交信号,信号物质分布于胸质内,胞核阴性。上述结果表明大白鼠颌下腺能合成GnRH受体,颌下腺产生的GnRH可作用于颌上腺的靶细胞,参与颌下腺生理功能的调节。     

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.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

α-MELANOCYTE-STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α-melanocyte-stimulating hormone (α-MSH) in the entire hypothalamus as well as the amount of α-MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1-24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso-osmotic sucrose solution, and free granules and synaptosomes containing α-MSH were fractionated by means of continuous sucrose density gradient centrifugation. α-MSH was quantified by radioimmunoassay. The total amount of α-MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α-MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5-24-month-old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α-MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.     

TRIPHOSPHOINOSITIDE PHOSPHODIESTERASE IN DEVELOPING BRAIN OF THE RAT AND IN SUBCELLULAR FRACTIONS OF BRAIN

Abstract— An assay system for the measurement of triphosphoinositide phosphodiesterase in homogenates of rat brain is described. With triphosphoinositide (TPI) as substrate, and in the presence of 0·1 m -KCI and saturating amounts of diethyl ether, the activity of phosphodiesterase in myelinated brain was 400–500 μmoles of TPI hydrolysed per g wet wt. per hr. One quarter of the adult level of the enzyme was present in rat brain one day after birth, with the remainder being added prior to and during the early stages of myelination. On subfractionation of brain homogenates, substantial activity of the enzyme was located in the soluble portion and in the paniculate fractions enriched in myelin and synaptosomes. The enzyme associated with the particulate fractions could not be detached from the membranes by any of several methods employed. There was a rough correlation between distribution of phosphodiesterase and that of 5'-nucleotidase, an enzyme associated with plasma membrane in a number of tissues. Some implications of the results are discussed.     

SYNTHESIS OF GANGLIOSIDES DURING DEVELOPMENT AND ITS RELATION TO THE QUANTITATIVE CHANGES OF SUBCELLULAR PARTICLES OF RAT BRAIN

Abstract— Different subcellular fractions prepared from developing rat cerebrum were quantified by determinations of dried weights and protein contents. It was found that there is an increase of 2–2·5 times in the weights of synaptosomes and mitochondria/g of tissue during the first 20 days of extrauterine life.
A period of fast synthesis of gangliosides, with a similar pattern of increase for the different subcellular particles, was found during the first 15 days of life by study of the incorporation of injected d -[1-¹⁴C]glucosamine in rats at different stages of development. Attempts to find out if gangliosides from a certain fraction are precursors of those of other fractions indicated that in all fractions the gangliosides increase independently of the other fractions. It is concluded that the enzymic systems of synthesis of these glycolipids are present in vivo in all the fractions considered.     

东北虎促卵泡激素和促黄体激素基因的克隆及其序列分析

本研究首次从东北虎（Panthea tigris altaica)脑垂体提取总RNA,利用RT－PCR技术扩增出东北虎垂体促性腺激素α亚基,促卵泡激素（FSH）β亚基和促黄体激素（LH）β亚基的编码区序列,并进行克隆,测序和比较分析。结果表明,其α亚基,FSHβ亚基,LHβ亚基基因的开放阅读框分别为363bp,390bp和420bp,分别编码120,129和142氨基酸的前体蛋白。与已发表的人,牛,绵羊,猪,大鼠,小鼠等物种相应序列比较,无论在核苷酸水平,还是在氨基酸水平都显示出较高的同源性（64．7％－96．6％）,其中与猪的同源性最高（86％－96．6％）。东北虎的基因序列还具有其明显的特异性,首次发现LHβ亚基cDNA编码的前体蛋白在信号肽部分比其它物种相应序列多一个亮氨酸残基（Leu)。     

促黄体素释放激素类似物(LH-RH-A)对妊娠大鼠子宫代谢的直接作用

本实验结果表明,胚泡着床点对~3H-尿嘧啶和~3H-亮氨酸的摄取明显高于非着床点子宫部位。LH-RH-A可显著抑制胚泡着床点对这两种同位素的摄取;且对非着床子宫组织也有抑制作用,但抑制程度较弱。进一步证实,着床的胚泡确能产生一种或几种因子,对子宫内膜的分化起重要作用;同时证实,LH-RH-A可通过对RNA和蛋白质合成的抑制作用而直接影响妊娠大鼠子宫的代谢,对胚泡着床点部位的影响尤为明显。     

间断低氧对大鼠下丘脑超微结构及前增食欲素水平的影响

目的探讨睡眠中间断低氧对大鼠下丘脑前增食欲素及受体水平的影响以及下丘脑超微结构的变化。方法大鼠分成对照组、间断低氧组和持续低氧组,分别给予吸入空气,持续低氧和间断低氧气体,并在实验开始后1d、3d、1w和4w应用RT-PCR方法测定大鼠下丘脑前增食欲素及受体水平,分析其间的变化关系,电镜观察下丘脑的超微结构变化。结果与对照组和持续低氧组比较,间断低氧4w后大鼠下丘脑前增食欲素mRNA水平明显降低,受体水平升高,但在持续低氧和对照组之间无明显差异。在低氧后1d、3d、7d后大鼠下丘脑前增食欲素mRNA降低,受体水平升高,在4w后,持续低氧组则接近正常。急性持续低氧大鼠超微结构变化更严重,而慢性间断低氧变化更持久。结论慢性间断低氧可以引起下丘脑前增食欲素下降及受体水平升高,急性持续低氧也可引起上述变化,而慢性持续低氧未引起增食欲素改变;慢性间断低氧大鼠下丘脑超微结构表现为严重而持久的变化。     

THE EFFECT OF LIMB ISCHAEMIA ON THE TURNOVER OF NORADRENALINE IN THE HYPOTHALAMUS AND BRAIN STEM OF THE RAT

The effect of ischaemic limb injury on the turnover of noradrenaline in the hypothalamus and brain stem has been studied in rats. There are theoretical reasons for thinking that these regions are activated in trauma and previous work showed that during limb-ischaemia the concentration of noradrenaline in the hypothalamus decreased by 27 per cent. The tourniquets were applied to both hind-limbs 1 h after the injection of [¹⁴C]-tyrosine when the labelling of the noradrenaline was maximal. During 4 h limb ischaemia the endogenous tyrosine concentration in the plasma decreased while that in the hypothalamus first rose and then fell. Changes in a similar direction in the brain stem were not statistically significant. Limb ischaemia did not affect the decline in the specific activity of the plasma or tissue tyrosine. It was concluded that the injury increased the utilization of tyrosine by the body. During the 4 h bilateral hind-limb ischaemia the rate of decline of [¹⁴C]noradrenaline was significantly increased in the brain stem but not in the hypothalamus. Conditions in the brain stem were sufficiently close to ‘steady-state’ to be able to conclude that the injury increased the metabolism of noradrenaline in the brain stem. Conditions in the hypothalamus were too complicated for definite conclusions to be drawn. The possible reasons for this and the limitations of this method for studying noradrenaline turnover are discussed.     

大鼠肺内NOS阳性神经及其来源的研究

应用还原型尼可酰胺腺嘌呤二核苷酸磷酸硫辛酰胺脱氢酶（ＮＡＤＰＨｄ）组织化学技术和ＨＲＰ逆行追踪与ＮＡＤＰＨｄ组化结合法,对大鼠肺内一氧化氮合酶（ＮＯＳ）阳性神经及其来源进行了研究。结果证实,肺内支气管和肺血管存在不同密度的ＮＯＳ阳性神经纤维,肺内神经节中存在少量ＮＯＳ阳性神经细胞。用ＨＲＰ与ＮＡＤＰＨｄ结合法,在迷走神经结状节和胸。。脊神经节中发现ＨＲＰ－ＮＯＳ双标记细胞,说明肺内ＮＯＳ神经纤维部分来自结状节和脊神经节,并对此进行了讨论。     

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.     

不饱和脂肪酸在牛、大鼠脑亚细胞组分中分布的比较

采用密度梯度离心等方法,从牛和大鼠脑组织中分别分离得到髓磷脂（A）、突触囊（B）、轻突触体（C）、重突触体（D）、线粒体（E）和微粒体（F）六个亚细胞组分。通过气相色谱－质谱联用手段和内标法测定了不饱和脂肪酸在各个亚细胞组分中的含量,结果表明牛和大鼠脑不饱和脂肪酸的亚细胞分布存在明显的差异。这种差异可能与动物种属、动物生活习惯等因素有关。