Some characteristics of mitochondrial monoamine oxidase activity in eggs of carp (Cyprinus carpio) and rainbow trout (Salmo gairdneri)

1. Monoamine oxidase (MAO) activity towards tryptamine, 5-hydroxytryptamine (5-HT) and phenylethylamine (PEA) has been measured in mitochondria isolated from carp and trout eggs. 2. In carp eggs all the tested substrates are metabolized and the highest affinity is found with tryptamine. In trout eggs a consistent level of MAO activity is obtained using tryptamine. 3. The inhibition dose-response curves of clorgyline and deprenyl indicate that both in carp and trout eggs there is only one form of mitochondrial MAO, distinct from MAO A and B which have been described in vertebrate tissues. 4. Both in carp and trout egg mitochondria a semicarbazide-sensitive amine oxidase is not involved in the deamination of the used substrates. 5. MAO found in carp and trout eggs might be involved in metabolism of some neurotransmitter monoamines during early developmental stages.     

Mitochondrial monoamine oxidase activity during preneural developmental stages of Bufo bufo

Monoamine oxidase specific activities against PEA and 5-HT have been measured in mitochondria isolated from early embryos of Bufo bufo. During the early development up to the neural fold stage, MAO activity undergoes a continuous decrease that is more evident when PEA is used as the substrate. The inhibition patterns of deprenyl and clorgyline demonstrate that, at the neural fold stage, both type A and B MAO are present. Both in eggs and embryos MAO type A activity appears slightly more sensitive to the inhibitory effect of various concentrations (0.1-2 M) of the denaturing agent urea.     

Changes in monoamine oxidase activity by mitochondria isolated from late embryos of Bufo bufo

1. Monoamine oxidase activity has been assayed in mitochondria isolated from post-neural embryos (stages 14-25) of Bufo bufo, using 5-HT and PEA as substrates. 2. Mitochondria isolated from stages 19 to 25 show an increasing ability in deaminating monoamines, PEA being metabolized at a higher level with respect to 5-HT. 3. At all the examined stages 5-HT is metabolized by an enzyme corresponding to MAO A, while PEA, from stage 19, is largely deaminated by a semicarbazide-sensitive amine oxidase (SSAO). 4. The effect of Triton X-100 on MAO A activity appears remarkably different in mitochondria isolated from embryos at stages 14 and 25 respectively.     

Monoamine oxidase activity in hepatopancreas of Octopus vulgaris

1. Monoamine oxidase activity has been studied in hepatopancreas of Octopus vulgaris using 5-HT and PEA as substrates.2. Time courses of MAO activity against 5-HT and PEA show that the enzyme has higher affinity for PEA than for 5-HT.3. MAO activity against 5-HT appears more sensitive than MAO activity against PEA, to variations of the temperature (range 17–67°C).4. The inhibition curves obtained with clorgyline and deprenyl indicate that MAO activity is due to a single form of the enzyme, not corresponding to type A and type B MAO.5. Semicarbazide 10⁻⁴ M does not affect the deamination of 5-HT and PEA, demonstrating that a semicarbazide-sensitive amine oxidase is not involved in this process.     

Monoamine oxidase and semicarbazide sensitive amine oxidase activities in toad liver mitochondria

1. The deamination of 5-HT and PEA has been assayed by a radiochemical method in mitochondria isolated from toad liver. 2. Time courses of 5-HT and PEA deamination indicate that when PEA is used as the substrate, higher specific activities are obtained. 3. 5-HT is deaminated by MAO A and partially by a SSAO-like enzyme. 4. PEA is deaminated exclusively by SSAO and, MAO B activity, at least under the adopted experimental conditions, is not detectable.     

Developmental changes in the activity and substrate specificities of mouse brain monoamine oxidase

Developmental changes in monoamine oxidase (MAO) activity in the mouse brain were investigated with the substrates -phenylethylamine (PEA), tryptamine, and 5-hydroxytryptamine (5-HT). In the newborn brain, MAO activity towards PEA was found to be much lower than the adult and to be inhibited by clorgyline in a double-sigmoidal fashion. The inhibition curve shifted to a single-sigmoidal pattern with age. MAO activity towards 5-HT as substrate was inhibited by 90% and in a single-sigmoidal manner by clorgyline throughout the postnatal life. Lineweaver-Burk plots with PEA as substrate presented two linear lines (apparentK _m: 28.6 and 4.1 M) for the newborn and one line (apparentK _m: 11.4 M) for the adult, respectively. The plot with highK _m value for the newborn brain disappeared in a clorgyline-treated preparation. These findings suggest that age-dependent alterations in the ratio of MAO-A/MAO-B activity affect the substrate specificity of the enzyme.     

Specificity of endogenous substrates for types A and B monoamine oxidase in rat striatum

Abstract: Studies were designed to evaluate specificity of the transmitter amines serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA), as well as the trace amines p -tyramine ( p -TA) and β -phenylethylamine (PEA) for types A and B monoamine oxidase (MAO) in rat striatum. 5-HT was found to be a specific substrate for the type A enzyme. However, the specificity of PEA for the type B enzyme was found to be concentration-dependent. When low concentrations of PEA and 5-HT were used to measure type B and type A activities, respectively, both clorgyline and deprenyl were highly selective for the sensitive form of MAO in vivo. However, as the concentration of PEA was increased, the type B inhibitor deprenyl became less effective in preventing deamination of PEA. Conversely, the type A inhibitor clorgyline became more effective in this regard. Kinetic analysis following selective in vivo inhibition showed PEA deamination by both forms of MAO with a 13-fold greater affinity for the type B enzyme. In vivo dose-response curves obtained with the common substrates DA and p -TA showed approximately 20% deamination by the B enzyme. Kinetic values for DA and p -TA deamination in in vivo -treated tissue possessing only type A or type B MAO activity, revealed a 2.5-fold greater affinity for the type A enzyme. These studies show the importance of concentration on substrate specificity in striatal tissue. The results obtained characterize the common substrate properties of DA and p -TA as well as of PEA in rat striatum. In addition, the presence of regional specificity for 5-HT deamination by only type A MAO is demonstrated.     

Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside

Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.     

Monoamino-oxidase activity in rat pineal gland. Histochemical studies

Monoamine-oxidase (MAO) activity was detected in rat pineal gland with dopamine, 5-hydroxytryptamine (5-HT), norepinephrine and tryptamine as substrates, and nitroblue tetrazolium salt as electron acceptor. Pinealocytes stained deeply when 5-HT was the substrate. Dopamine and tryptamine substrates gave similar patterns, with moderate activity in the pinealocytes. Norepinephrine reactivity was detected in the nerve-endings.     

Liquid chromatographic and tandem mass spectrometric assay for evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors: application of biomarkers in drug discovery

A simple and selective assay for the evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors was developed through the simultaneous determination of endogenous 5-hydroxy tryptamine, 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophane, and 2-phenethylamine (PEA) in rat brain using liquid chromatography-tandem mass spectrometry (LC/MS/MS). These analytes were separated on a Zorbax SB-C18 column using a gradient elution with acetonitrile and 0.2% formic acid and detected on an electrospray ionization mass spectrometer in positive-ion multiple-reaction-monitoring mode. The susceptibility and variability of these analytes as potential biomarkers in response to MAO inhibition in vivo were evaluated after application to three MAO inhibitors, tranylcypromine, clorgyline, and pargyline. A dramatic increase (about 40-fold) in PEA brain level and a decrease in 5-HIAA by more than 90% were observed after administration of 15 mg/kg of the nonselective MAO inhibitor tranylcypromine. As expected, the brain level of PEA escalated to about 6-fold, while the 5-HIAA level remained unchanged following a dose of the MAO B inhibitor pargyline at 2mg/kg. In contrast, the brain level of 5-HIAA reduced by approximately 53%, but the PEA level was unaffected following the same dose of the MAO A inhibitor clorgyline. The results indicated that 5-HIAA and PEA were susceptible and effective biomarkers in the rat brain in response to MAO A and B inhibition, respectively. The LC/MS/MS method is useful not only for the determination of inhibitory potency but also for the differentiation of the selectivity of a MAO inhibitor against rat brain MAO A and B in vivo.     

Characterization of monoamine oxidase activity present in human granulocytes and lymphocytes

The characterization of monoamine oxidase (MAO) activity in lymphocytes and granulocytes was studied by using cells prepared from human blood. The specific activities of the enzyme towards beta-phenylethylamine (PEA), benzylamine (Bz), tyramine (TYR) and 5-hydroxytryptamine (5-HT) were found to be 5-times higher in lymphocytes than in granulocytes. The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by the lack of effect of semicarbazide on the benzylamine oxidation. The presence of MAO-B was corroborated by the inhibition of PEA oxidation with nanomolar deprenyl concentrations and by inhibition of TYR oxidation with high clorgyline concentrations, as well as by the simple sigmoid curve obtained in both cases. These results, together with the substrate preferences, suggest that the MAO activity of human granulocytes and lymphocytes is predominantly of the B form. For each fraction the kinetic constants were determined towards PEA, TYR and Bz as substrates. The Km values were similar for both cellular samples, whereas the Vmax values were higher in lymphocytes than in granulocytes. MAO-B was titrated with [3H]pargyline in order to find out the number of active sites. The corresponding molecular concentration, Kcat values and turnover number showed the presence of related enzymes in human granulocytes and lymphocytes.     

Monoamine Oxidase Activities in Catfish (Parasilurus Asotus) Tissues

Abstract

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For both tissues, the V_max values were highest with 5-HT and lowest with PEA. The K_m value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 μM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.     

Inhibition of rat brainstem monoamine oxidase activity by CGP 6085 A

CGP 6085 A [4-(5,6-dimethyl-2-benzofuranyl)piperidine] HCl, a known serotonin inhibitor, also inhibits rat brainstem monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in both in vivo and in vitro experiments. Serotonin (5-HT) deamination by MAO-A is inhibited 35% at a dose of 100 mg/kg i.p. in vivo. Similar experiments show a maximal 20% decrease in phenylethylamine (PEA) deamination by MAO-B at a dosage of 30 mg/kg i.p. Over the range of 0.1 to 10 mg/kg i.p., CGP 6085 A decreases 5-HIAA levels in the brainstem. This in vivo inhibition of MAO activity is confirmed by in vitro experiments. In vitro studies in rat brainstem mitochondrial preparations show a dose-dependent, reversible, inhibition of MAO using tyramine as the substrate for the enzyme reaction. With an in vitro IC50 of 2-3 microM, the potency of CGP 6085 A is comparable to pargyline.     

Substrate Selectivity of Type A and Type B Monoamine Oxidase in Rat Brain

Abstract: Use of the irreversible inhibitors clorgyline and deprenyl showed that rat brain mitochondria contain type A and type B monoamine oxidase (MAO). Tyramine is a substrate for both types of MAO, whereas serotonin is a preferential substrate for type A MAO. In contrast to MAO in other tissues, type A MAO in brain tissue oxidizes β-phenylethylamine (PEA) at high concentrations (0.5 and 1.0 mM). The proportions of type A and type B MAO activities in the mitochondria estimated from the double-sigmoidal inhibition curves of tyramine oxidation were about 70:30 irrespective of the concentration of tyramine. With PEA as substrate, the ratios of type A to type B activities were found to increase from low values at low concentrations to about 1 at 0.5-1.0 mM-PEA, and even higher at further increased concentrations of PEA. At very low (0.01 mM) and high (10.0 mM) concentrations of PEA, single-sigmoidal curves were obtained; with the high PEA concentration the activity was highly sensitive to clorgyline, whereas with the low concentration it was highly sensitive to deprenyl. In deprenyl-pretreated mitochondrial preparations, all the remaining activity towards 0.5-1.0 mM-PEA was shown to be highly sensitive to clorgyline, demonstrating that this activity was indeed due to oxidation by type A MAO. The opposite result was obtained with deprenyl as inhibitor of clorgyline-pretreated preparations, demonstrating that PEA at this concentration was also oxidized by type B MAO in rat brain mitochondria. The K₃ values of type A and type B MAO for PEA were significantly different. On Lineweaver-Burk analysis, plots with PEA as substrate for type A MAO in a deprenyl-treated preparation were linear over a wide concentration range, whereas those for type B MAO in a clorgyline-treated preparation were not linear, but showed substrate inhibition at higher concentrations of the substrate. It is concluded from the present findings that the effect of the substrate concentration must be considered in studies on the characteristics of multiple forms of MAO in various organs and species.     

Monoamine oxidase A mediates iodotyrosine formation induced by monoamines in bovine thyroid particulate fraction

Monoamines are able to increase the thyroid iodine organification in vitro. A predominance of the A form of monoamine oxidase (MAO) has been previously demonstrated to exist in bovine thyroid tissue. In the present study we have investigated the form of MAO that could be involved in the iodotyrosine formation induced by tyramine, 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PEA) in a bovine thyroid subcellular fraction. The relative capacity of these monoamines to generate H2O2 and to incorporate iodine into tyrosine has also been studied. The MAO A inhibitor clorgyline (10(-9) M) produced a strong inhibition on the iodotyrosine formation induced by tyramine, 5-HT and PEA. In contrast, only a slight reduction was observed with deprenyl as MAO B inhibitor. Among the three monoamines, tyramine produced the highest H2O2 generation and iodotyrosine formation. The lowest Km value obtained was for 5-HT and the highest for PEA. Regarding the Vmax, the lowest value was for 5-HT and the highest for tyramine. The amount of iodine incorporated to tyrosine was not equivalent to the H2O2 generated by the monoamines nor to that exogenously added. Our results indicate that in bovine thyroid tissue mainly the A form of MAO is involved in the monoamine metabolism.     

Antagonism between long acting Monoamine Oxidase Inhibitors (MAOI) and MD780515, a new specific and reversible MAOI

The inhibition of type A and B monoamine oxidase (MAO A and B) in rat brain, liver and heart by MD780515, 3-[4-(3 cyanophenylmethoxy) phenyl]-5-(methoxymethyl)-2-oxazolidinone, has been investigated ex vivo with 5-hydroxytryptamine (5-HT) and β-phenylethylamine (PEA) as substrates. MAO A was strongly inhibited for four hours after oral administration of 10 mg/kg MD780515 (maximum inhibition : 72%, 86% and 83% in brain, liver and heart respectively. In contrast, in heart where PEA is deaminated by type A MAO, the predominant form of MAO in that tissue, the inhibition was 68% 30 minutes after administration of the compound. In all cases, MAO activities reached control values 24 hours after drug administration (10 mg/kg), whereas some inhibitory activity was still present 24 hours after oral administration of higher doses. The strong MAO A inhibition (68 to 83%) remaining in the three tissues 24 hours after oral administration of clorgyline (5 mg/kg) was completely removed by pretreatment with MD780515 (10 mg/kg). In the same conditions, MD780515 protected against the inhibition (53%) by clorgyline of PEA deamination in heart. Oral pretreatment with increasing doses of MD780515 (2.6 to 84 mg/kg) gradually removed brain MAO A inhibition caused by clorgyline (92%, 28.2 mg/kg) or tranylcypromine (88%, 4.8 mg/kg), the complete removal being observed at the dose of 21 mg/kg of MD780515 for clorgyline, and at 42 mg/kg for tranylcypromine. Inhibition of brain MAO B by tranylcypromine (96%) was not modified by pretreatment with the same range of oral doses of MD780515. The results are consistent with a specific and reversible inhibition of MAO A activity by MD780515 which can protect against long acting MAO A inhibitory effects of clorgyline and tranylcypromine. MD780515 enhances the selectivity of tranylcypromine.     

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.