Effect of centrophenoxine, piracetam and aniracetam on the monoamine oxidase activity in different brain structures of rats

In vitro studies of effects of some nootropic drugs (centrophenoxine, piracetam and aniracetam) on monoamine oxidase (MAO) activity in the rat striatum and hypothalamus, using tyramine, serotonin and beta-phenylethylamine as substrates, were carried out. At all concentrations used (5.10(-5)-1.10(-3) M) centrophenoxine inhibited total MAO, MAO A and MAO B in both brain structures. Piracetam activated striatal and hypothalamic total MAO, hypothalamic MAO A and MAO B but exerted a pronounced inhibitory effect on MAO A and MAO B activity in the striatum. Aniracetam inhibited total MAO and MAO A in both brain structures but activated striatal and hypothalamic MAO B. The different effects of centrophenoxine, piracetam and aniracetam on MAO activity in the brain structures support the view for the independent mode of action of nootropic drugs in spite of their similar molecular and metabolic activity.     

Problems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations. Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data.

There are significant discrepancies in the literature concerning the concentration of monoamine oxidase A (MAO A) from a number of tissue sources. Therefore, we compared the two principal techniques that have been used for quantitation of MAO A protein concentration: (1) titration of the enzyme with the MAO A-selective inhibitor clorgyline, and (2) saturation of the enzyme with [3H]-pargyline followed by immunoprecipitation with an MAO A-specific monoclonal antibody. To determine which of the two techniques was likely to yield more reliable values for MAO A, MAO A protein concentrations in the same preparations were determined by quantitative immunoblotting. [3H]Pargyline binding and quantitative immunoblotting yielded comparable values which were markedly lower than those obtained by titration of MAO A with unlabeled clorgyline. Therefore, clorgyline titration can seriously overestimate the concentration of MAO A protein in mitochondrial preparations. Since many literature values for the molecular activity of MAO A have relied upon enzyme concentrations determined by clorgyline binding, we reevaluated the molecular activities of MAO A and B for five important substrates. The ratio, MAO A molecular activity:MAO B molecular activity decreased in the order: serotonin (35:1) greater than tryptamine (12:1) greater than tyramine (3.3:1) greater than dopamine (2.4:1) greater than benzylamine (1:23). No comparable ratio was determined for beta-phenylethylamine because of its previously described substrate inhibition of MAO B, although it is oxidized faster by MAO B over a wide range of concentrations. Comparison of molecular activities and Km values for MAO A and B showed that with the exception of benzylamine and beta-phenylethylamine, MAO A oxidizes the other tested substrates faster than MAO B over a wide range of concentrations. Therefore, measured ratios of MAO A:MAO B activity are generally greater than the ratios of MAO A:MAO B molecules in the preparations.     

对—羟基苯甲醛和对—羟基苯甲酸对单胺氧化酶活性的影响

小鼠灌服对一羟基苯甲醛(PHBAD)和对一羟基苯甲酸(PHBZA)能明显抑制脑和肝组织的单胺氧化酶(MAO)活性,且对MAO—B的抑制作用强于MAO—A。PHBAD(200～400mg·kg~(-1)对小鼠脑MAO—B抑制作用强于PHBZA。体外实验证明,二者对MAO—B的抑制作用随浓度增加而明显增强。另外,PHBAD对MAO—B呈竞争型抑制;对MAO—A呈混合型抑制;而PHBZA对MAO-B、MAO—A均呈混合型抑制。     

Distribution of type A and type B monoamine oxidase activities in rat and chicken skeletal muscle and nerves

The monoamine oxidase (MAO) activities and proportions of type A and type B MAO in rat and chicken skeletal muscles, sciatic nerves, phrenic nerves, brachial plexi, spinal cords and motor cortices were studied. Skeletal muscle contained mainly or exclusively type A MAO. The MAO in neural tissue of both species was 30–95 per cent type A, with chicken neural tissue tending to have more type B MAO than the rat. There was no evidence of a correlation between the total MAO activity of particular nerves and the MAO activity of the skeletal muscles they innervate. The MAO activity of the skeletal muscles did not correlate with their frequencies of contraction.     

Monoamine oxidase in single nerve cell bodies from locus coeruleus of the rat. A microgasometric study

The magnetic diver microgasometer was used for determination of MAO activity in single nerve cell bodies isolated from the locus coeruleus of the rat. Tyramine was used as a substrate. Both molecular forms of MAO, MAO A and MAO B, are present in single nerve cell as shown by clorgyline, a selective inhibitor of MAO A molecular form. The activity of MAO in nerve cell bodies from locus coeruleus was compared to the activities in seven other types of nerve cells.     

Preferential inhibition of the B-form of monoamine oxidase in the liver of rats given 3'-methyl-4-dimethylaminoazobenzene in the diet

A and B-form monoamine oxidase (MAO) activities were measured in the liver of rats maintained with a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). A-form MAO activity was similar to the control value throughout the feeding periods with serotonin as substrate. In contrast, B-form MAO activity decreased rapidly and the level of MAO activity was maintained at about 30% with beta-phenylethylamine (beta-PEA) as substrate. 3'-Me-DAB feeding did not cause any changes in MAO activity in the brain of rats. A single administration of 3'-Me-DAB (100 mg/kg p.o.) failed to alter A and B-form MAO activities for up to 4 days after its administration. The mechanism of inhibition of B-form MAO activity in rat liver mitochondria by 3'-Me-DAB was investigated. The inhibition of 3'-Me-DAB of B-form MAO activity, in vitro, was competitive and reversible. There was no difference in the apparent Michaelis constant toward beta-PEA between control and 3'-Me-DAB fed rats. B-form MAO in rat liver mitochondria was titrated with (-)deprenyl; this compound is selective to and an irreversible inhibitor of B-form MAO. The content of B-form MAO in liver mitochondria of rats fed 3'-Me-DAB for 3 weeks was decreased to about 60% of the control level.     

Thermal inactivation of mouse brain monoamine oxidase type A and type B

Mouse brain monoamine oxidase (MAO) type A and type B were incubated at 54 degrees C and samples removed for up to 60 min, and remaining MAO activity was determined. Total MAO activity, type A activity and type B activity all disappeared, presumably due to thermal denaturation, in a time-dependent fashion. The rate of disappearance of MAO type B was faster than that of type A both at pH 7.4 and at pH 9.2, though both types denatured faster at the higher pH compared to the lower pH.     

酶标仪比色法测定单胺氧化酶活性

目的建立用酶标仪比色测定单胺氧化酶A(monoamine oxidase A,MAO A)和MAO B活性的方法。方法以大鼠肝组织匀浆作酶原,分别以血清素(5-HT)和苄胺作为MAO A和MAO B底物,以2,4-二硝基苯肼(DNPH)和氢氧化钠为显色剂,在450 nm处用酶标仪比色测定。结果该方法能快速、灵敏、稳定、选择性地测定MAO活性。结论建立了以酶标仪比色法测定MAO活性的方法用于筛选药物。     

Platelet monoamine oxidase activity in first-degree relatives of schizophrenic patients

Platelet monoamine oxidase (MAO) is under genetic control. A lower MAO activity in chronic schizophrenia has repeatedly been reported, and it has been suggested that reduced activity of this enzyme reflects an increased vulnerability to schizophrenia. To test this hypothesis platelet MAO was determined in 65 first-degree relatives of 22 schizophrenic index patients and in matched healthy controls. No difference in mean activity between the two samples could be detected, suggesting that reduced MAO activity in schizophrenia is more likely to be a phenomenon secondary to the disease. A significant parent-offspring correlation of MAO activities was obtained.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Increase of brain endogenous monoamine oxidase inhibitory activity (tribulin) in experimental audiogenic seizures in rats: evidence for a monoamine oxidase A inhibiting component of tribulin.

Brain tribulin activity in rats with an inherited predisposition to audiogenic epilepsy was studied after seizures of different intensity were induced by an electric bell. Weak seizures (from 0 to 2 arbitrary units) did not produce any changes in endogenous inhibitory activity towards either monoamine oxidase (MAO) A or B. Moderate seizures were characterized by increases in both MAO A and MAO B inhibitory activity (up to 1.9-fold). Complete tonic epileptiform seizures with total areflexia (4 arbitrary units) induced further augmentation (up to 2.5-fold) of MAO A but not of MAO B inhibitory activity. This dissociation between the two inhibitory activities points to the existence of a separate MAO A-inhibiting component of brain tribulin which is different from isatin.     

Development of benzylamine oxidase and monoamine oxidase A and B in man

We describe the widespread distribution of the enzymes benzylamine oxidase (BzAO) and monoamine oxidase A and B (MAO A, MAO B) in human tissues at three stages of development: fetal, neonatal and adult. Relatively low activity of each is present in fetal tissues, but the specific activities of BzAO and MAO B in fetal liver are similar to those in the adult, suggesting that these enzyme systems are functionally mature in the liver at 20 weeks gestation. Specific activity of BzAO in the lung, twice as high in the adult as in the fetus, reaches adult value at birth. In fetal brain, lung, aorta and digestive tract, MAO A emerges before MAO B. Estimation of total activity developed by tissue or organ sheds a new light on the importance of each enzyme in body economy and shows skeletal muscle to be by far the most active whole body source of MAO B, whilst the liver has the highest total and specific activity of MAO A. A similar approach clearly demonstrates that BzAO is essentially a tissue rather than a plasma enzyme, which tends to predominate in blood vessel walls.     

Characterization of rat skeletal muscle monoamine oxidase.

Optimal conditions for deamination of 5-hydroxytryptamine in rat skeletal muscle were determined. The presence of monoamine oxidase (MAO) A and MAO B isozymes was demonstrated by the use of tyramine (a substrate of both forms), specific substrates (serotonin and benzylamine), and specific inhibitors (clorgyline and deprenyl) of MAO A and B respectively. A 6.5:3.5 ratio of MAO A to B was found using a whole muscle homogenate, while a 7.5:2.5 ratio was found with isolated mitochondria. Thermal inactivation studies demonstrated that skeletal muscle MAO A is more susceptible to heat inactivation than MAO B. The approximate proportion of muscle homogenate MAO which is present in sympathetic nerves was found to be 18 per cent, as determined by treating rats with 6-hydroxydopamine and quantifying the decrease in activity. Significant inhibition of MAO activity was observed after administration in vivo of the MAO inhibitors pargyline, tranylcypromine and harmaline.     

Effects of zinc ion on type A monoamine oxidase in monkey brain mitochondria

The effects of ZnSO(4) on types A and B monoamine oxidase (MAO) isozymes in monkey brain mitochondria were investigated, in vitro. Type A MAO activity in monkey brain decreased to about 50% with 1 microM ZnSO(4) using serotonin as a substrate, and this inhibition was proportional to the concentration of ZnSO(4). ZnSO(4) had no effect, however, on type B MAO activity in monkey brain using beta-phenylethylamine as a substrate. The inhibition by ZnSO(4) of type A MAO activity was competitive and reversible. ZnSO(4) did not inhibit either type A or type B MAO activity in rat brain mitochondria. Almost similar results were also obtained when ZnCl(2) was used, in vitro. These results indicate that the inhibiting action of zinc ion differs depending on animal species and organ. Type A MAO in monkey brain mitochondria was highly sensitive to zinc ion, while type B activity was less sensitive.     

Further studies on the synthesis of A-form monoamine oxidase

The relation between precursors and restoration of A-form MAO activity in rat liver after administration of clorgyline to rats was investigated by measuring the rates of recovery of A-form MAO activity after treatment with the inhibitor. The half-lives of mitochondrial and microsomal A-form MAO were estimated as 3.5 and 2.0 days, respectively. MAO activity and the amount of MAO molecules were completely restored within 14 days. However the values attained did not exceed the control values in a period of 14 days. Clorgyline plus cycloheximide or chloramphenicol did not prevent the recovery of MAO activity in the microsomes, but did not delay the appearance of enzyme activity in the mitochondria. A and B-form-like MAO were also observed in the microsomal and supernatant fractions, with clorgyline as inhibitor. These results suggest that the microsomal enzyme is a precursor of the mitochondrial enzyme, that the levels of A-form and B-form MAO are regulated genetically, and that the two forms of MAO may be synthesized separately.     

β-Phenylethylamine and benzylamine as substrates for human monoamine oxidase A: A source of some anomalies?

Monoamine oxidase (MAO) A predominates both in human placenta and lung. With 5-hydroxytryptamine (5-HT), β-phenylethylamine (PEA) and benzylamine (Bz) as substrates and clorgyline and deprenyl, respectively, as selective MAO A and B inhibitors, their activity pattern has been defined and compared with that of human liver. PEA had a much higher V_max with placental MAO A than did Bz; it behaved largely as an A substrate in placenta, and partly as an A substrate in lung. At commonly used substrate concentrations, deamination of Bz (sensitive to 10^?7 M deprenyl) was a better indicator of MAO B activity than deamination of PEA. The divergence between PEA and Bz as MAO A and B substrates may be one reason for some of the apparent discrepancies in the behaviour of MAO A and B noted in a variety of tissues in the literature.However, Bz reacts with benzylamine oxidase (BzAO) as well as MAO B. Depending on the tissue, deprenyl-resistant Bz activity may indicate the presence of BzAO rather than MAO A. As there is a widespread distribution of BzAO in man and rat, BzAO should be considered among the alternatives of enzyme activity when Bz is used as substrate.     

Differences in the structures of monoamine oxidases A and B in rat clonal cell lines

[3H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in two rat cell lines were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [3H]Pargyline was bound to MAO A and B in a crude mitochondrial fraction from rat hepatoma cell line MH1C1 and to MAO A in a mitochondrial fraction from rat glioma line C6. Specific radiolabeling of proteins associated with type A or B activity in the hepatoma samples was achieved by incubation with selective B or A inhibitors, respectively, prior to [3H]pargyline binding. Following [3H]pargyline binding, the samples were solubilized by heating in sodium dodecyl sulfate (SDS) in the presence of a reducing agent. SDS-PAGE of [3H]pargyline bound samples revealed a radiolabeled protein band of apparent molecular weight (mol. wt) 63,000 daltons associated exclusively with MAO A activity and a band of apparent mol. wt 60,000 associated exclusively with MAO B activity. Furthermore, when SDS-solubilized, [3H]pargyline-labeled MAO A and B proteins from these cell lines were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [3H]pargyline-labeled peptides were obtained. These findings indicate that the A and B forms of MAO activity are associated with enzyme molecules of different primary covalent structures determined by different gene loci.     

Characterization and quantitation of monoamine oxidases A and B in mitochondria from human placenta

Monoamine oxidases (MAOs) A and B, flavin-containing enzymes found in the outer mitochondrial membrane, oxidize many important biogenic and xenobiotic amines. The two enzymes are expressed in many tissues, with some tissues containing primarily one form and others containing both. Although MAO in placental mitochondria is widely reported to be type A, some investigators have reported low levels of MAO B activity as well. Because placenta is considered the preferred source for purification of type A MAO, we have reinvestigated placental MAO by immunoblotting with monoclonal antibodies and active site labeling with the MAO-specific ligand [3H]pargyline. We have confirmed that placental mitochondrial preparations contain MAO A and low but significant MAO B catalytic activity, as judged by accepted pharmacological criteria (deprenyl-sensitive beta-phenylethylamine and benzylamine oxidation). Immunoblotting revealed polypeptides of sizes expected for both MAO A and B subunits in preparations of placental mitochondria, as well as in preparations of MAO A purified extensively from placenta by partitioning between dextran and polyethylene glycol polymers and chromatography on DEAE-Sepharose CL-6B. Both MAO A and B active sites could be quantitated in placenta by labeling mitochondrial preparations with the MAO-specific affinity ligand [3H] pargyline, followed by immunoprecipitation with MAO A- and MAO B-specific monoclonal antibodies. These results indicate that MAO B activity and protein is consistently present in mitochondrial preparations of human placenta.     

串果藤中甘草素和异甘草素对大鼠单胺氧化酶的体外抑制作用(英文)

目的:研究串果藤中甘草素和异甘草素体外对单胺氧化酶A型和B型的抑制作用.方法:根据不同的离心速度制备大鼠全脑粗线粒体作为单胺氧化酶的酶源;分别以5-羟基[侧链-2-~(14)C]色胺肌酸硫酸盐([~(14)C]5-HT)和2-苯基[1-~(14)C]乙基胺盐酸盐([~(14)C]β-PEA)为单胺氧化酶A型和B型放射性底物,用液体闪烁技术,研究甘草素和异甘草素酶抑制作用和抑制类型.结果:甘草素和异甘草素对单胺氧化酶A型和B型均具有抑制作用,呈良好的量效关系,对单胺氧化酶A型的IC_(50)(95％的可信限)分别为32(26-36)μmol/L和13.9(12.8-15.6)μmol/L,对单胺氧化酶B型的IC_(50)值分别为104.6(89.0-118.9)μmol/L和47.2(39.5-54.5)μmol/L.酶抑制特征曲线显示甘草素和异甘草素对单胺氧化酶A型呈非竞争性抑制,K_i值分别为31.5μmol/L和14.3μmol/L,而对单胺氧化酶B型呈混合竞争性抑制,K_i值分别为164.7μmol/L和62.2μmol/L,K_I值分别为15.2μmol/L和9.3μmol/L.结论:甘草素和异甘草素体外对单胺氧化酶A型呈非竞争性抑制作用,对单胺氧化酶B型呈混合竞争性抑制作用.     

Use of a monoclonal antibody for comparative studies of monoamine oxidase B in mitochondrial extracts of human brain and peripheral tissues

Monoamine oxidase B (MAO B; EC 1.4.3.4) activity in detergent extracts of mitochondria from autopsy brain (gray matter and medulla), liver, lung, and kidney from a single individual and from pooled, human platelets could be immunoprecipitated by a monoclonal anti-human platelet MAO B antibody (MAO-1C2) in combination with appropriate secondary reagents. MAO A activity, which was detected in brain, liver, lung, and kidney, was not immunoprecipitated under the same conditions. All MAO B-containing extracts, regardless of tissue source, inhibited immunoprecipitation of [3H]pargyline-labeled human platelet MAO, and the shapes of the inhibition curves were identical. The concentration of immunologically detectable MAO B protein in the extracts was estimated from immunoprecipitation competition data by reference to a standard curve relating observed inhibition of immunoprecipitation to the concentration of catalytically active platelet MAO added (estimated from [3H]pargyline binding data). MAO B protein concentrations measured by this radioimmunoassay were similar to concentrations of active MAO B as measured by pargyline binding. These results demonstrate that in the brain and peripheral tissues studied, molecules with MAO B activity share a unique antigenic determinant and similar catalytic efficiency. They also extend previous observations that MAO B molecules extracted from mitochondria bear an antigenic determinant which is not present on MAO A molecules. These results demonstrate the validity of a new competitive radioimmunoassay for active plus inactive MAO B concentration in human platelet extracts and extracts of mitochondria from human tissues. This radioimmunoassay should complement [3H]pargyline binding assays and enzyme activity assays in studies designed to clarify the mechanisms of genetic, disease, and treatment factors which lead to differences in MAO B function among individuals.     

Specific changes in type A and B monoamine oxidase activity in different tissues of hypophysectomized rats

MAO activity was tested in four organs of young male rats 16 days after hypophysectomy. Five monoamines were used as substrates. In the adrenal glands and the thyroid gland MAO activity towards the substrates preferably deaminated by MAO-A was severely decreased. In the adrenal glands, but not in the thyroid gland, the effect of hypophysectomy could be simulated with dexamethasone, a steroid which inhibits ACTH secretion. A significant rise in MAO activity towards substrates for MAO-A and MAO-B occurred in the heart of hypophysectomized rats. There was little change in the MAO activity of whole brain homogenates after hypophysectomy.