1,6-Diaminohexane contributes to the hexamethylene bisacetamide-induced erythroid differentiation pathway by stimulating Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores and promoting Ca2+ influx

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.     

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.     

肿瘤细胞诱导分化剂NADAH在硅胶载体上的固定化及其与DNA作用的研究

1976年Reuben等[1]发现六亚甲基二乙酰胺（hexamethylenebisacetallllde,HMBA）在5×10-3mol/L浓度可诱导99％以上的Friend红白血病细胞分化．已报道HMBA在体外可引起动物多种实体瘤和白血病细胞系的分化［2］．阐明HMBA诱导肿瘤细胞分化的机制有着重要意义．HMBA去掉一个乙酸基后单乙酸己二胺（N-acetyl-diallllnohexaneNADAH）：CH3CONHCHZCHZCHZCHZCHZCHZNHZ,具有与HMBA几乎同样诱导肿瘤细胞分化的活性［3,'」,由于NADAH有一个活泼基因NHZ,为固相化研究其诱导肿瘤细胞分化机理提供了可能。通过两步合成将…     

Catabolites produced by the deacetylation of hexamethylenebisacetamide play a key role in murine erythroleukaemic-cell differentiation.

N-Acetyl-1,6-diaminohexane and 1,6-diaminohexane, formed by deacetylation of the inducer hexamethylenebisacetamide (HMBA), are shown to accumulate rapidly inside murine erythroleukaemic cells. The appearance of these molecules preceded the differentiation-associated changes in intracellular polyamines. A quantitative relationship was observed between the accumulation of these molecules and the changes in intracellular polyamines. In the absence of HMBA, exogenous N-acetyl-1,6-diaminohexane was able not only to cause changes in polyamine biosynthesis, but also to induce the complete differentiation process. These results imply that these catabolites of HMBA are directly responsible for the changes in polyamine biosynthesis and probably also for initiating other events regulatory for the differentiation of these cells.     

Comparative study of substrate and inhibitory specificity of monoamine oxidase of squid optic ganglia

Comparative study of substrate specificity of monoamine oxidase (MAO) of optic ganglia of the Pacific squid Todarodes pacificus and the Commander squid Berryteuthis magister has been carried out. The enzyme of the Pacific squid, unlike that of the Commander squid, has been established to be able to deaminate not only tyramine, tryptamine, serotonin, benzylamine, and β-phenylethylamine, but also histamine-substrate of diamine oxidase (DAO). In relation to all studied substrates, the MAO activity of optic ganglia of T. pacificus is several times higher as compared with that of B. magister. In the case of deamination of serotonin this difference was the highest and amounted to 5 times. Semicarbazide, the classic DAO inhibitor, at a concentration of 10 mM did not inhibit catalytic activity of both studied enzymes. The substrate-inhibitory analysis with use of deprenyl and clorgyline, specific inhibitors of different MAO forms, indicates homogeneity of the enzyme of the Pacific squid and heterogeneity of the Commander squid enzyme whose composition seems to contain at least two MAO forms. There are obtained quantitative differences in substrate specificity and reaction capability with respect to the inhibitors clorgylin and deprenyl for MAO of optic ganglia of the studied squid species. These differences probably can be explained by significant differences in the evolutionary level of these biological species.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia

D-amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K(i) = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase,product of a susceptibility gene for schizophrenia

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K_i = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues

We have previously reported that carp (Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or beta-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.     

Striatal dopamine metabolism in monoamine oxidase B-deficient mice: a brain dialysis study

We have studied striatal dopamine (DA) metabolism in monoamine oxidase (MAO) B-deficient mice using brain microdialysis. Baseline DA levels were similar in wild-type and knock-out (KO) mice. Administration of a selective MAO A inhibitor, clorgyline (2 mg/kg), increased DA levels and decreased levels of its metabolites in all mice, but a selective MAO B inhibitor, l-deprenyl (1 mg/ kg), had no effect. Administration of 10 and 50 mg/kg L-DOPA, the precursor of DA, increased the levels of DA similarly in wild-type and KO mice. The highest dose of L-DOPA (100 mg/kg) produced a larger increase in DA in KO than wild-type mice. This difference was abolished by pretreating wild-type mice with l-deprenyl. These results suggest that in mice, DA is only metabolized by MAO A under basal conditions and by both MAO A and B at high concentrations. This is in contrast to the rat, where DA is always metabolized by MAO A regardless of concentration.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus <Emphasis Type="Italic">Bathypolypus arcticus</Emphasis>

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, b-phenylethylamine, while, on the other hand, it deaminates histamine and does not deaminate putrescine-classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs for the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine-irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.     

Monoamine Oxidase Activity in the Hepatopancreas of the Kamchatka Crab <Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>: a Substrate–Inhibitor Specificity

A study of substrate–inhibitor specificity of mitochondrial monoamine oxidase (MAO) in the hepatopancreas of the adult Kamchatka crab Paralithodes camtschaticus revealed specific catalytic properties of the enzyme. On the one hand, crab hepatopancreas MAO, like its classical hepatic counterpart, can deaminate tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine and N-methylhistamine but shows no sensitivity to 10 mM semicarbazide. On the other hand, MAO deaminates histamine but not putrescine, two classical diamine oxidase (DAO) substrates. It was established that MAO activity was several times higher toward benzylamine, β-phenylethylamine and N-methylhistamine than toward serotonin and noradrenalin. MAO was also found to be almost 500 times more sensitive to its selective inhibitor deprenyl than to chlorogilyn. A substrate–inhibitory analysis with the use of deprenyl and chloroginyl provides an indirect evidence for the existence of a sole MAO molecular form in the Kamchatka crab hepatopancreas.     

Monoamine oxidase in adult Ascaridia galli

Monoamine oxidase (MAO), catalysing oxidative deamination of biogenic monoamines, has been detected in adult Ascaridia galli. MAO was present in mitochondria and deaminated noradrenaline at the maximal rate, although serotonin, adrenaline, tyramine and dopamine were also degraded but more slowly. Of the organs studied, the body wall, female reproductive organ and intestine, the body wall (containing neuronal structures) showed highest MAO activity. Km value for chick ascarid mitochondrial MAO using tyramine as substrate was 1.66 X 10(-3) M and it was most active at 2.5 mM tyramine concentration, pH 7.5 and 40 degrees C. MAO of A. galli appeared to be thermolabile as nearly 80% of its activity was lost when the incubation temperature was increased 5 degrees above optimum.     

Mode of action of formamidine pesticides: an evaluation of monoamine oxidase as the target.

The ability of formamidine pesticide, chlordimeform (N'-(4-chloro-o-toyl)-N,N-dimethylformamidine) (CDM), and several of its major metabolites to inhibit monoamine oxidase (MAO) in mouse tissues in vitro and in vivo was examined, and related to the hypothesis that inhibition of MAO is responsible for the lethal effects of CDM. CDM was a readily reversible inhibitor of MAO of medium potency as were most of its metabolites. However, the hydrolysis product, N-formyl-4-chloro-o-toludine (CT) was a significantly more potent reversible inhibitor. A comparison of MAO from brain, liver, and intestine showed no marked variations in their sensitivity to these inhibitors. Greater inhibitory potency was found using Type A substrates (5-hydroxytryptamine) than Type B substrates (beta-phenylethylamine). The activity of MAO in vivo after pretreatment of mice with CDM or its metabolites was assessed in liver and intestine by measuring the amount of [14C] tryptamine which still survived 5 min after an intraperitoneal injection. Established inhibitors of MAO gave appropriate results with this method. CDM also increased tryptamine recoveries but only at does which caused mortality, and then to a lesser extent than MAO inhibitors such as tranylcypromine, pheniprazine, and harmaline at sub lethal doses. For this reason, and in view of the lack of correlation of toxicity to MAO-inhibitory potency among CDM and its metabolites, and because the symptoms of poisoning are inappropriate, it is concluded that MAO inhibition is not an important factor in the acute lethality of CDM.     

Influence of some inhibitors of histamine metabolism on the gastric secretion

Influence of some inhibitors of histamine metabolism on the gastric secretion. Acta Physiol. Pol., 1977, 28 (6): 515-520. The influence of inhibitors of histamine metabolism on histamine (H) and Nalpha Nalpha-dimethylhistamine (NDMH) stimulated gastric secretion was studied in guinea-pigs and cats. Inhibitors of monoamine oxidase (MAO) and diamine oxidase (DAO): N-oxide diacetylaminopyridine (AAP) and N-oxide 2 aminopyridine (AP) increased HCI secretion in the gastric juice after H and NDMH. Inhibitors of N-methyl transferase: amodiaquine (A) and quinacrine (Q) increased HC1 secretion in the gastric juice after H but not after NDMH. The lack of action of A and Q on NDMH-stimulated gastric secretion suggests, that in guinea-pig and cat NDMH is not methylated additionally at the imidazole ring and therefore, it is a stronger gastric secretagogue than histamine itself.     

Propranolol inhibits rat brain monamine oxidase.

Propranolol and its d-isomer inhibit monoamine oxidase (MAO) from the brain of the rat. The I₅₀ for each was 260 μM, compared to a value of 23 μM for pargyline. The I₅₀ for the local anesthetic procaine was 22 μM in this system. Practolol, a β-blocker that is not a local anesthetic, had only weak activity at 1 mM. Levels of serotonin (5-HT) were increased in the cerebral cortex of rats by treatment with d,1-propranolol (12.5–50 mg/kg), whereas levels of 5-hydroxyindoleacetic acid (5-HIAA) were decreased. Levels of 5-HT were also increased by treatment with similar doses of d-propranolol, but not by treatment with practolol. It was concluded that propranolol inhibits MAO and the metabolism of 5-HT by a mechanism unrelated to blockade of β-adrenergic receptors and that this activity may be related to the local anesthetic properties of this drug.     

Plasma amine oxidases in Wilson's disease

Plasma Mono- and Diamine-Oxidase activities (MAO and DAO), two copper containing enzymes, were estimated in 5 patients with Wilson's disease, without treatment and during D-Penicillamine treatment. Ceruloplasmin and “free” copper plasma levels were simultaneously measured. MAO was elevated in all cases, while DAO was within normal limits.D-Penicillamine administration did not result in significant reductions of these enzyme activities. It is likely that alterations of copper metabolism induced by Wilson's disease and by D-Penicillamine administration do not affect the activity of MAO or DAO. The increase in MAO activity in Wilson's disease probably results from the hepatic fibrosis.     

The metabolism and binding of catecholamines by the hepatic microsomal mixed-function oxidase of the rat.

Noradrenaline and adrenaline were metabolized by an NADPH- and oxygen-dependent process located within the hepatic microsomal fraction of the rat. Metabolism was inhibited by CO and compound SKF 525A, but not by pargyline, an inhibitor of monoamine oxidase, or by 3,4-dimethoxy-5-hydroxybenzoic acid, an inhibitor of catechol O-methyltransferase. It is concluded that the enzyme system responsible for the metabolism of the catecholamines was the microsomal mixed-function oxidase. The Km for noradrenaline was 2.4 mM and for adrenaline 1.0 mM, and V 15.6 and 3.6 nmol/min per mg of microsomal protein respectively. Both catecholamines bound to the microsomal fraction, producing a type II spectral change, with a Ks for noradrenaline of 0.9 mM and for adrenaline of 1.0 mM, and showed other characteristics of type II compounds by inhibited the reduction of cytochrome P-450 by NADPH and exhibiting an enhanced metabolism in the presence of acetone. The major product of catecholamine metabolism was an as yet unidentified alkali-labile compound, which did not correspond to any of the recognized catecholamine metabolites.     

Inhibition and inactivation of monoamine oxidase by 3-amino-1-phenyl-prop-1-enes.

We have previously shown that E-3-amino-1-phenyl-prop-1-ene (E-cinnamylamine) is readily oxidised by monoamine oxidase (MAO) type B from either rat or bovine liver (Williams et al. (1988), Biochem. J. 256, 411-415) in each case producing a non-linear progress curve which was attributed to inhibition by the reaction product E-cinnamaldehyde. We have now found that although this aldehyde inhibits MAO B competitively (Ki 0.017 mM) this cannot account for the inhibitory process, since during a 60 min incubation with the substrate (0.5 mM; Km, 0.074 mM) more than 95% inhibition of MAO B was observed and the concentration of aldehyde had reached approx. 0.025 mM. Inhibition was relieved either by dialysis or dilution of inhibited samples. The activity of MAO A from rat liver was largely unaffected by E-cinnamylamine. Oxidation of N-methyl-E-cinnamylamine and its Z-isomer by MAO B produced progress curves similar to that obtained with the primary amine, but in these cases inhibition was not reversed either by dilution or dialysis. Partition ratios for the pair of N-methyl isomers with bovine MAO B were calculated to be 1640 (E-isomer) and 1430 (Z-isomer). The time-dependent inhibition process for all three amines obeyed pseudo-first-order kinetics. A tritiated form of N-methyl-E-cinnamylamine, incubated with MAO B from bovine liver, resulted in incorporation of radioactivity into the enzyme. This labelling was stable to dialysis and to SDS-PAGE.