THE CHARACTERIZATIO OF MONOAMINE OXIDASE IN CULTURED MAMMALIAN CELLS

The expression of monoamine oxidase (MAO) in a variety of mammalian cells has been investigated employing tryptamine as substrate. The enzyme present in those cell lines having sufficient activity for detailed analysis exhibited a monophasic response to the inhibitor clorgyline. On this basis the cell lines examined were found to express only A, or only B, type activity. Hypoxanthineguanine phosphoribosyl transferase (HGPRT) deficient derivatives of both MAO type A, or MAOtype B, expressing cells were examined. The HGPRT status of the cells appeared to have little influence on the expression of the enzyme.     

SUBSTRATE AND INHIBITOR-RELATED CHARACTERISTICS OF MONOAMINE OXIDASE IN C6 RAT GLIAL CELLS

Cultured C6 rat glial cells preferentially deaminated 5-hydroxytryptamine, tryptamine, dopamine and tyramine in comparison to phenylethylamine and benzylamine. Deamination of all substrates was uniformly sensitive to inhibition by clorgyline and relatively insensitive to deprenyl. These data together with the observations of simple sigmoid curves for the inhibition of tyramine deamination by both inhibitors suggest that C6 glial cells contain mainly monoamine oxidase type A, which previously had been suggested to be primarily an intraneuronal MAO type. As these findings are in agreement with other studies of brain MA0 activity in mitochondria separated from neuronal vs glial cell preparations, they help explain why MA0 activity measured with some substrates may be little affected by lesions or by drugs producing nerve ending degeneration.     

OCCURRENCE AND PROPERTIES OF MONOAMINE OXIDASE IN ADRENERGIC NEURONS

—Monoamine oxidase activity of peripheral organs of various species has been examined after surgical, chemical and immunological sympathectomy to assess the proportion of enzyme activity in adrenergic neurons and in extraneuronal cells. Significant falls in monoamine oxidase activity of vas deferens, submaxillary gland, iris and spleen were seen after sympathetic denervation although not in heart, small intestine and kidney. It was suggested that a correlation exists between the extent of the fall in monoamine oxidase activity after sympathectomy and the density of sympathetic innervation of the control organ. Studies of monoamine oxidase activity in vas deferens after inhibition with clorgyline suggested multiple forms of monoamine oxidase. Differences in inhibitor sensitivity, substrate specificity and thermal inactivation of monoamine oxidase in normal and denervated vas deferens were found and it was suggested that differences exist in the properties of the neuronal and extraneuronal monoamine oxidase.     

PURIFICATION AND PROPERTIES OF MITOCHONDRIAL MONOAMINE OXIDASE IN BEEF BRAIN

Abstract— Monoamine oxidase was purified approximately 40-fold from beef brain mitochondria. The purification procedure involved extraction with a non-ionic detergent (Nonion NS-210) after heat treatment, ammonium sulphate fractionation, chromatographies on DEAE-cellulose and Sepharose 6B, and a continuous flow electrophoresis. A major component (enzyme 1) with a higher specific activity and a minor component (enzyme 2) with a lower specific activity were separated. Properties of both enzymes towards kynuramine including pH-optimum and K_m values were similar, but the enzyme 1 had the higher specific activity towards tyramine whereas that of enzyme 2 was towards normetane-phrine. Fluorescence spectra indicated that the enzyme 1 is a flavoprotein. Copper was not detected, and copper chelating agents did not inhibit the enzyme. p -Chloromercuribenzoate and JV-ethylmaleimide inhibited the enzyme, indicating the presence of the essential SH-groups.     

NONEXISTENCE OF A TYPE C MONOAMINE OXIDASE IN RAT BRAIN

Abstract— The possible existence of type C MAO, distinct from type A and type B, in circumventricular structures of rat brain was examined by histological studies on the inhibitory effects of clorgyline. a preferential type A MAO inhibitor and deprenyl, a preferential type B inhibitor, on enzyme. Brain slices were preincubated with the inhibitors and then incubated with 5-HT, the substrate for type A MAO, and stained for MAO activity. Deposits of the product formazan were detected in circumventricular structures of slices of brain preincubated with clorgyline and deprenyl at concentrations of 10^-7–10^-4m at room temperature for 5 min. When the slices were preincubated with either of these inhibitors at room temperature for 60 min, strong activity was observed in this region, whereas when they were preincubated with either 10^-5m -clorgyline or 10^-5m -deprenyl for 20 and 30 min at 37°C, no MAO activity was seen in any region of the brain. Thus, at the higher preincubation temperature, lower concentrations of each inhibitor and a shorter preincubation period were required for inhibition of the enzyme. Preincubation for 60 min at 37°C with a combination of 10^-7m -clorgyline and 10^-8m -deprenyl did not inhibit the enzyme in the circumventricular region completely, but at the same temperature, concentrations of 10^-7m of both inhibitors inhibited the enzyme completely in 10min, Thus the effects of the inhibitors are synergistic. These results indicate that the inhibitory effects of the two inhibitors on the enzyme in circumventricular structures of the brain is time- and temperature-dependent. Moreover, the activity seems to be sensitive to deprenyl even when 5-HT is used as substrate. The results do not support the idea of the existence of type C MAO, distinct from type A and type B MAO.     

MONOAMINE OXIDASE ACTIVITY IN NORMAL and LESCH-NYHAN FIBROBLASTS

Monoamine oxidase (MAO) activity was studied in cultured skin fibroblasts from 10 Lesch-Nyhan patients, a Lesch-Nyhan variant and 11 controls matched for age. sex and race. Activity (predominantly type A) was measured in cell homogenates using tryptamine as the substrate. For each line activity varied with the conditions of culture. Activity increased 3-10 fold as cultures went from logarithmic to stationary phase of growth. When cultures were confluent, activity was lowered by frequent feedings or the use of fresh medium and serum. Activity for each line remained fairly stable during successive passages, but rose 3-8 fold as cultures became senescent. When comparing activity between control and Lesch-Nyhan lines, cells were cultured under standardized conditions. The mean value of MAO activity in Lesch-Nyhan lines was approximately one fourth of the mean activity in control lines (P < 0.012), In the control population, the distribution of activity appeared to be bimodal. Activities in the Lesch-Nyhan lines fell completely within the lower portion of the control distribution. Cells from a Lesch-Nyhan patient who lacked several of the neurologic symptoms of the disease (including self-mutilation) had an MAO activity 6 fold greater than the control mean. although his hypoxanthine phosphoribosyltransferase activity was <3% of control levels. It appears that: (1) MAO activity is low in fibroblasts from typical Lesch-Nyhan patients: (2) the severity of neurologic symptoms may be correlated with levels of MAO activity: and (3) some interaction between purine and catecholamine metabolism can affect nerve function.     

DEVELOPMENTAL CHANGES OF CHOLINESTERASES AND MONOAMINE OXIDASE IN CHICK EMBRYO SPINAL AND SYMPATHETIC GANGLIA

Abstract— Total cholinesterase, acetylcholinesterase, (AChE) and monoamine oxidase (MAO) activity and protein content were determined throughout the embryonic life of the chick in spinal and sympathetic ganglia. The greatest part of total cholinesterase activity was due to AChE.
AChE and MAO activity increased in both spinal and sympathetic ganglia very similarly from the 6th to the 12th day of incubation; from this day on a significant divergence occurred, mainly owing to a steady fall in spinal ganglion AChE, which decreased to approximately one tenth of the maximum value. The ratio of MAO activity in sympathetic and spinal ganglia increased from the 8th day onwards and approached 5·0 at hatching. The ratio between sympathetic and spinal ganglia, for AChE, choline acetylase (ChAc) and MAO activity, suggests a relationship between the maturation of the synapse in the sympathetic ganglia and the maximal activity of these enzymes.     

MULTIPLE FORMS OF MONOAMINE OXIDASE IN DEVELOPING BRAIN: TISSUE AND SUBSTRATE SPECIFICITIES

Brains, hearts and livers from newborn and adult rats were assayed for monoamine oxidase activity using gel electrophoretic techniques. The results suggest that each of the tissues possesses multiple forms (isoenzymes) of monoamine oxidase and that these forms are different for the various tissues. Further, the forms of monoamine oxidase in the neonatal tissues differ from those in the corresponding adult tissue. These different forms of monoamine oxidase have different substrate specificities. Using 5-hydroxy[¹⁴C]tryptamine as substrate, we have demonstrated that the monoamine oxidase patterns appearing on the gel do indeed possess monoamine oxidase activity.     

老龄大鼠心肌单胺氧化酶的研究

本文比较研究了年轻鼠和老龄鼠心肌线粒体MAO的活力变化,及MAO活力对温度的依赖性。结果表示老龄心肌MAO对底物的亲合性略有下降,但酶活力增加了近十倍。在12—43℃范围内绘制的MAO活力的Arrhenius图提示老龄鼠线粒体膜脂的物理性状发生了改变。     

EFFECT OF OESTRADIOL ON TURNOVER OF TYPE A MONOAMINE OXIDASE IN BRAIN

Abstract— Administration of oestrogen (oestradiol-17β or oestradiol-17β-benzoate) to ovariectomized (OVX) rats for 1–4 weeks results in an approx 30% decrease in the activity of monoamine oxidase (MAO) in the basomedial-hypothalamus (BM-Hyp) and corticomedial-amygdala (CM-Amy) but not in cerebral cortex. Further investigation shows that (1) decreased MAO activity in the BM-Hyp and CM-Amy occurs only in Type A MAO (serotonin as substrate) and does not occur in Type B MAO (phenylethylamine as substrate); (2) decreased MAO activity does not occur when a single large dose of oestrogen is given i. v. or when homogenates from oestrogen treated rats are mixed with homogenates from OVX rats suggesting that direct enzyme inhibition is not responsible for the change in activity; (3) oestrogen administration to OVX rats increases the rate constant of degradation for MAO in BM-Hyp and CM-Amy but not in cerebral cortex as determined in turnover studies using pargyline, an irreversible inhibitor of MAO. The increased rate of degradation results in shorter half lives ( t 1/2) for MAO in the BM-Hyp and CM-Amy of oestrogen treated rats. In OVX rats the t 1/2 is 9.8 days in BM-Hyp and 12.7 days in CM-Amy. Oestrogen administration results in a t 1/2 of 7.6 days in BM-Hyp and 7.8 days in CM-Amy. The possible relationship between oestrogen dependent decreased MAO activity and estrogen dependent lordosis behavior is discussed.     

LOWERED MONOAMINE OXIDASE ACTIVITY IN BRAINS FROM ALCOHOLIC SUICIDES

—Monoamine oxidase (MAO) activity in the brains of 15 suicides, of whom 8 were alcoholics, was compared to a control material of 20 individuals without known mental disorder. At autopsy 13 different parts of the brain were macroscopicaily dissected out and the MAO activity in the samples estimated with β-phenylethylamine and tryptamine as substrates. The MAO activity in all parts of the brain investigated was found to be significantly lower in the alcoholic suicides as compared to the controls, while there was no significant difference between the non-alcoholic suicides and controls. Different variables which might have influenced the MAO activity were investigated. There was no significant correlation between age and tryptamine-oxidizing activity, but a positive correlation between age and β-phenylethylamine-oxidizing activity was found. There was also a significant difference between the series in the time lapse between death and autopsy and in the time during which the dead body was kept at room temperature. However, neither of these variables could explain the differences between the series. The results thus demonstrate a connection between low MAO activity in the brain and suicidal behaviour among alcoholics.     

大豆磷脂对老龄大鼠单胺氧化酶,血小板,动脉内膜的影响

本文观察了饲料添加剂大豆磷脂对老龄大鼠脑组织单胺氧化酶(MAO)活力、血小板聚集功能及主动脉内膜的影响。结果表示喂大豆磷脂24月龄大鼠组的大脑皮层、小脑、海马的MAO活力较对照组高,接近3月龄组;二磷酸腺苷(ADP)诱导血小板释放三磷酸腺苷(ATP)的量高于3月龄组,低于对照组;主动脉内膜扫描电镜观察内皮细胞轮廓清晰可见,有少数桥样结构,表面未见斑块样附着物,对照组则呈粥样硬化形态特征。上述结果提示,大豆磷脂作为饲料添加剂对某些衰老现象有明显的改善。     

CALCIUM FLUXES IN CULTURED AND BULK ISOLATED NEURONAL AND GLIAL CELLS

Abstract— The influx and efflux of ⁴⁵Ca has been studied in cultured human glioma and mouse neuroblastoma cells and in isolated fractions enriched in synaptosomes, neuronal and astrocytic perikarya from rabbit brain.
The uptake of ⁴⁵Ca was somewhat more efficient in glioma compared to neuroblastoma cells, whereas there was little difference in the rate of ⁴⁵Ca uptake by isolated glial cells and neuronal perikarya. Isolated synaptosomes showed the highest rate of ⁴⁵Ca accumulation. An increase of K concentration to 50 m m in the medium, with a corresponding lowering of Na, stimulated both glioma and glial as well as synaptosomal ⁴⁵Ca uptake more markedly than the uptake by neuroblastoma cells and neuronal perikarya. Lowering the Na concentration and replacing it by choline had no effect on the cultured cells and astrocytes. Na-free media caused massive stimulation of ⁴⁵Ca influx in all fractions and cells tested.
The efflux of ⁴⁵Ca was studied after preloading of cells. Three phases could be resolved from the desaturation curves. All cells had nearly similar half-lives for ⁴⁵Ca efflux under standard conditions. Pulses of media containing 50 m m -K stimulated ⁴⁵Ca efflux from glioma cells and astrocytes more efficiently than from neuroblastoma cells, neuronal perikarya and synaptosomes. The stimulated release was exclusively seen in Ca-containing media in experiments with the cultured cells and in Ca-free media in experiments with cell perikarya. The effect of transmitter pulses on the release of ⁴⁵Ca was examined in a limited series. Acetylcholine and isoproterenol were found to stimulate ⁴⁵Ca release more actively from glia than from neurons.     

次黄嘌呤与黄嘌呤氧化酶引起的培养神经细胞的损伤以及缺糖和谷氨酸盐的影响

在外源性次黄嘌呤和黄嘌呤氧化酶的作用下,培养神经细胞发生肿胀、破碎和K~ 丢失等损伤性变化。当细胞外葡萄糖由5mmol/L降至2.5mmol/L及Ommol/L时,K~ 丢失分别增加1.4倍及7.8倍。若同时加入谷氨酸盐1mmol/L或N-甲基-D-天冬氨酸(NMDA)0.4mmol/L,K~ 丢失增高3倍。提高Mg~(2 )浓度至2.5mmol/L或加入DL-2-氨基-5-磷戊酸0.5mmol/L均可阻断谷氨酸盐对自由基损伤的增强作用。提示脑缺血,缺氧时,兴奋性氨基酸通过激动NMDA受体,使Ca~(2 )内流增加,从而加重自由基损伤。     

胡萝卜培养细胞的盐适应蛋白

近年来植物离体培养细胞盐适应蛋白的发现以及盐影响下植物信使 RNA 体外转译产物的研究,为耐盐分子机理的研究和耐盐基因的鉴定和克隆开辟了道路。烟草细胞在适应1—2.5%NaCl 之后,细胞蛋白质合成发生变化,特别是26kD 的蛋白质大量积累。体内标记实验表明,细胞从不加 NaCl 的培养基转到加有1%