Phospholipid dependency of carp brain and liver mitochondrial monoamine oxidase

1. The effects of lipid-protein interactions on carp brain and liver mitochondrial MAO with respect to substrate and inhibitor preference, thermostability and Arrhenius parameters were studied and compared.2. Treatment with phospholipase A₂, C or D decreased MAO activities towards 5-hydroxytryptamine (5-HT), β-phenylethylamine and tyramine similarly, accompanied by great changes in their apparent affinities for MAO, but not by changes in V_max values.3. Minimum phospholipid binding to mitochondria might be essential for enzyme activity.4. Among these activities, 5-HT deamination was the most sensitive to the changes in mitochondrial phospholipids and bulk lipid phase transition (fluidity).5. Sensitivity of MAO to clorgyline or l-deprenyl was not affected by these phospholipase treatments.6. Of the phospholipids tested, only phosphatidylinositol significantly activated MAO activity towards 5-HT in both intact and phospholipase-treated mitochondria.     

Substrate-dependent activation energy of the reaction catalyzed by monoamine oxidase

The effect of temperature on the deamination of 5-hydroxytryptamine, tyramine, and phenethylamine by monoamine oxidase (MAO) of human placenta, beef liver, and rat liver has been studied. Both MAO A and MAO B activities are influenced by the lipid-phase transition and, in some cases, another type of transition. The estimates of activation energy (E_act) for the deamination of 5-hydroxytryptamine, phenethylamine, tyramine, dopamine, and pentylamine at 5–20 °C show that a given substrate is associated with a particular value irrespective of the source of MAO acting upon it. The substrate dependence of E_act is explained by the differences in lipophilicity of the various substrates. The interaction of enzyme and the lipids in the environment of its active site would differ with each substrate, and would give rise to different activated complexes, each corresponding to a given substrate. The E_act values are presumably related to these complexes, rather than to enzyme alone.     

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain

LRRK2 gain-of-function is considered a major cause of Parkinson’s disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.     

Zinc,copper, and zinc- or copper-dependent enzymes in human hypertension

Imbalance of zinc and copper status has been hypothesized in human hypertension. A case-control study was carried out to elucidate the possible relationship between zinc and copper status and essential hypertension. Thirty-one subjects affected by mild stable hypertension, pharmacologically untreated, were investigated together with 31 normotensive controls individually matched for sex, age, and smoking habits. Zinc and copper in serum and urine were measured, and serum activities of alkaline phosphatase (AP), lactic dehydrogenase (LDH), copper-zinc superoxide dismutase (Cu?Zn SOD), lysyl oxidase (LOX), and monoamine oxidase (MAO) were evaluated. No significant difference in serum and urine zinc and copper content as far as in serum activity of zinc (AP and LDH) or copper (Cu?Zn SOD, LOX, and MAO)-dependent enzymes was found between hypertensives and normotensives. Positive relationships were found in normotensives between serum and urine levels of zinc (r=0.577;p=0.001) and copper (r=0.394;p=0.028), and between serum copper and Cu?Zn SOD (r=0.534;p=0.002). In normotensives, diastolic blood pressure and serum zinc were positively related (r=0.370;p=0.041). In hypertensives, inverse correlations were observed between diastolic blood pressure and AP (r=?0.498;p=0.004) and Cu?Zn SOD (r=?0.452;p=0.011), and between systolic blood pressure and LOX (r=?0.385;p=0.033). Diastolic blood pressure was related to LDH inversely in hypertensives (r=?0.357;p=0.049) and positively in normotensives (r=0.457;p=0.010). In normotensives, diastolic blood pressure was inversely related with MAO (r=?0.360;p=0.046). These findings support the hypothesis that an imbalance of zinc and copper status might be involved in human hypertension.     

Phenylethanolamine is a specific substrate for type B monoamine oxidase

The characteristics of phenylethanolamine as both a competitive inhibitor and as a substrate for monoamine oxidase (MAO) were studied using rat brain and liver homogenates. Although phenylethanolamine, even at high concentrations (1 mM), produced minimal inhibition of MAO when serotonin (a substrate for type A MAO) was used as the substrate, it was a potent competitive inhibitor (K_i=11 μM) of the deamination of phenylethylamine (a substrate for type B MAO). When phenylethanolamine was used as a substrate, deprenyl, a selective inhibitor of type B MAO, was found to produce a single sigmoid inhibition curve at low concentrations of the inhibitor (pI₅₀=7.5). These results indicate that phenylethanolamine is a specific substrate for type B MAO. Identification of the products formed under the assay conditions show that phenylethanolamine is converted to both mandelic acid and phenylethylene glycol by liver homogenates but only to the latter, neutral metabolite by brain homogenates.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Design,synthesis and evaluation of 4′-OH-flurbiprofen-chalcone hybrids as potential multifunctional agents for Alzheimer’s disease treatment

A series of 4′-OH-flurbiprofen-chalcone hybrids were designed, synthesized and evaluated as potential multifunctional agents for the treatment of Alzheimer’s disease. The biological screening results indicated that most of these hybrids exhibited good multifunctional activities. Among them, compounds 7k and 7m demonstrated the best inhibitory effects on self-induced Aβ_1–42 aggregation (60.0% and 78.2%, respectively) and Cu²⁺-induced Aβ_1–42 aggregation (52.4% and 95.0%, respectively). Moreover, these two representative compounds also exhibited good antioxidant activities, MAO inhibitions, biometal chelating abilities and anti-neuroinflammatory activities in vitro. Furthermore, compound 7m displayed appropriate blood-brain barrier permeability. These multifunctional properties highlight compound 7k and 7m as promising candidates for further development of multi-functional drugs against AD.     

2,2′-dithienyl diselenide,an organoselenium compound,elicits antioxidant action and inhibits monoamine oxidase activity in vitro

Context: Organoselenium compounds have been described as antioxidant and neuroprotective agents.

Objective: To evaluate the antioxidant action of 2,2′-dithienyl diselenide (DTDS) and its effects in brain monoamine oxidase (MAO) activity in vitro.

Materials and methods: Assays for reactive species (RS), lipid peroxidation, protein oxidation, MAO A and B activities in rat brain homogenate as well as mimetic dehydroascorbate reductase and glutathione S-transferase activities were performed using DTDS (μM range).

Results: DTDS was effective in decreasing the levels of RS as well as lipid peroxidation induced by malonate, sodium nitroprusside or FeCl₂/EDTA and protein carbonyl in the rat brain homogenate. DTDS elicited dehydroascorbate reductase-like and glutathione S-transferase-like activities. DTDS was effective in inhibiting both MAO-A and MAO-B activities.

Discussion: The results demonstrated that DTDS is an antioxidant agent with non-selective inhibitory effect on MAO activity.

Conclusion: DTDS is a promising molecule to be evaluated in experimental models of neurological diseases.     

Chromone 3-phenylcarboxamides as potent and selective MAO-B inhibitors

Monoamine oxidase (MAO) is an enzyme, present in mammals in two isoforms MAO-A and MAO-B. These isoforms have a crucial role in neurotransmitters metabolism, representing an attractive drug target in the therapy of neurodegenerative diseases (MAO-B) and depression (MAO-A). In this context, our work has been focused on the discovery of new chemical entities (NCEs) for MAO inhibition, based on the development of chromone carboxamides. Chromone derivatives with a carboxamide function located in position 2- and 3- of the benzo-γ-pyrone core, (compounds 2-6 and 8-12) were synthesized, with moderate/good yields, by a one-pot condensation reaction using phosphonium salts as coupling reagents. The synthetic compounds were screened towards human MAO isoforms (hMAO) to evaluate their potency and selectivity. The chromone-3-carboxamides show high selectivity to hMAO-B, with compounds 9 and 12 displaying IC₅₀ values at nanomolar range.     

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in V_max of both MAO isoenzymes in serum-free medium, but no change in K_m. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.     

Chromone-2- and -3-carboxylic acids inhibit differently monoamine oxidases A and B

Chromone carboxylic acids were evaluated as human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitors. The biological data indicated that only chromone-3-carboxylic acid is a potent hMAO-B inhibitor, with a high degree of selectivity for hMAO-B compared to hMAO-A. Conversely the chromone-2-carboxylic acid resulted almost inactive against both MAO isoforms. Docking experiments were performed to elucidate the reasons of the different MAO IC₅₀ data and to explain the absence of activity versus selectivity, respectively.     

Comparative enzymologic study of catalytical properties of liver monoamine oxidases of sturgeon fish

We carried out the comparative study of the substrate and inhibitory specificity of liver monoamine oxidases (MAO) of the giant sturgeon Huso huso, the starred sturgeon Acipenser stellatus, the Persian sturgeon Acipenser persicus, and the Russian sturgeon Acipenser gueldenstaedtii. Results of the substrate-inhibitor analysis with use of inhibitors chlorgilin and deprenil, as well as five specific substrates indicate homogeneity of these enzymes. All studied MAO have the several orders higher sensitivity to chlorgilin than to deprenil, with essential interspecies differences being observed. There are determined kinetic parameters of enzymatic deamination (K _M and V) of tyramine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, and N-methylhistamine. All studied enzymes have been established to have the higher activity toward serotonin and noradrenalin-substrates of the MAO A form as compared with benzylamine, β-phenylethylamine, and N-methylhistamine-substrate of the mammalian MAO B form, the maximal activity being characteristic of the giant sturgeon.     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Development of selective and reversible pyrazoline based MAO-B inhibitors: virtual screening, synthesis and biological evaluation

In an effort to develop selective MAO (monoamine oxidase) B inhibitors, structure based virtual screening was initiated on an in-house library. Top 10 HITS were synthesized and evaluated for MAO (A and B) inhibitory activity, both against human and rat enzymes. All the compounds were found selective, reversible and active in nM range (100 times more potent than selegeline) towards MAO-B. Outstanding co-relation between predicted and experimental K_i values were observed.     

Propiconazole induced toxicological alterations in brain of freshwater fish Channa punctata Bloch

Fungicides are a class of pesticides which are used indiscriminately in large amounts and pose a serious threat to the environment. Propiconazole (PCZ) is a systemic foliar fungicide with a broad range of activity. The potential of this fungicide to induce toxicity has not been fully explored. The present study was designed to investigate the dose dependent neurotoxic effect of propiconazole (PCZ), with Channa punctata Bloch as a model organism. Effect of PCZ on the brain specific enzyme activity such as acetylcholinesterase (AChE), monoamine oxidase (MAO) and Na⁺-K⁺-ATPase was determined in the fish brain tissue exposed to sub-lethal concentrations (0.5 and 5 ppm) for 96 h. Also, levels of oxidative stress reflected by various enzymatic and non-enzymatic antioxidants were measured. Neurotransmitter (epinephrine) level was also assessed. PCZ exposure induced oxidative stress as reflected by the significant increase in fish brain lipid peroxidation and protein carbonyl content with decrease in reduced glutathione levels, as well as the significant inhibition of glutathione dependent metabolizing enzymes and CAT activities. In addition, AChE, MAO and Na⁺-K⁺-ATPase activities were significantly lowered along with reduction in epinephrine levels in PCZ exposed fishes than those of the control in a dose dependent manner. Also, histopathological alterations were observed in fish brain of the treated fishes. The results point towards the potential neurotoxicity in the fish caused by PCZ exposure but the application of these findings will need more detailed study before they can be established as special biomarkers for toxicity monitoring the aquatic environment.     

Monoamine oxidase A and B inhibiting effect and molecular modeling of some synthesized coumarin derivatives

New series of bioactive 7-oxycoumarin derivatives were synthesized and tested for their in vitro and in vivo monoamine oxidase (MAO) A and B inhibitory effect. In vitro studies revealed exceptionally potent and selective MAO-A inhibitors with K_i values on a picomolar range. The acetohydrazide (3b) and the dioxopyrrolidine derivative (7b) showed the most potent in vitro and in vivo MAO inhibition activity. Moreover, molecular modeling study of the synthesized compounds into MAO-A (PDB: 2Z5X) and MAO-B (PDB: 2XFN) binding sites exhibited direct correlation between AutoDock binding affinity and% inhibition MAO-A (pM) and MAO-B (μM). In addition, the results of in vivo MAO inhibiting properties (ED₅₀) of the tested compounds revealed better direct correlation.     

Comparative Analysis of Expression of Genes Encoding Enzymes of Catecholamine Catabolism and Renalase in Tissues of Normotensive and Hypertensive Rats

Comparative analysis of expression of genes encoding enzymes of catecholamine catabolism (monoamine oxidases A and B (MAO A and MAO B) and catechol-O-methyl transferase (COMT)) and renalase has been carried out in tissues of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Among investigated tissues the highest level of mRNA of genes encoding key enzymes of catecholamine catabolism (MAO A, MAO B, COMT) was found in the heart of WKY rats. In SHR the mRNA levels of these genes were lower (p < 0.05–0.01), however, no similar changes were observed in the tissues studied in dependence of hypertension. The relative mRNA levels of the studied genes normalized versus actin mRNA significantly varied. In heart and kidney the relative level of COMT mRNA significantly exceeded the relative levels of both MAO A mRNA and MAO B mRNA. In the brain differences in mRNAs of MAOA, MAOB, and COMT were less pronounced. However, in all examined tissue the renalase mRNA level was much (at least 10–20-fold) lower than any other mRNA studied. Taking into consideration known correlations between mRNAs and corresponding protein products reported in the literature for many genes these results suggest that in the case of any catalytic scenarios proposed or even proved for renalase this protein cannot contribute to catecholamine degradation. It is also unlikely that the products of the renalase reaction, β-NAD(P)⁺ and hydrogen peroxide, can exhibit a hypotensive effect due to low expression of the renalase encoding gene.     

Kinetic and structural analysis of the irreversible inhibition of human monoamine oxidases by ASS234, a multi-target compound designed for use in Alzheimer's disease

Monoamine oxidases (MAO) and cholinesterases are validated targets in the design of drugs for the treatment of Alzheimer's disease. The multi-target compound N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (ASS234), bearing the MAO-inhibiting propargyl group attached to a donepezil moiety that inhibits cholinesterases, retained activity against human acetyl- and butyryl-cholinesterases. The inhibition of MAO A and MAO B by ASS234 was characterized and compared to other known MAO inhibitors. ASS234 was almost as effective as clorgyline (k_inact/K_I = 3 × 10⁶ min^− 1 M^− 1) and was shown by structural studies to form the same N5 covalent adduct with the FAD cofactor.     

Synthesis and evaluation of 2-substituted 4(3H)-quinazolinone thioether derivatives as monoamine oxidase inhibitors

In the present study, a series of fourteen 2-mercapto-4(3H)-quinazolinone derivatives was synthesised and evaluated as potential inhibitors of the human monoamine oxidase (MAO) enzymes. Quinazolinone is the oxidised form of quinazoline, and although this class has not yet been extensively explored as MAO inhibitors, it has been shown to possess a wide variety of biological activities. Among the quinazolinone derivatives investigated, seven compounds (IC₅₀?<?1?µM) proved to be potent and specific MAO-B inhibitors, with the most potent inhibitor, 2-[(3-iodobenzyl)thio]quinazolin-4(3H)-one, exhibiting an IC₅₀ value of 0.142?μM. Further investigation showed that this inhibitor is a reversible and competitive inhibitor of MAO-B with a K_i value of 0.068?µM. None of the test compounds were MAO-A inhibitors. Analysis of the structure-activity relationships (SARs) for MAO-B inhibition shows that substitution on the C2 position of quinazolinone with a benzylthio moiety bearing a Cl, Br or I on the meta position yields the most potent inhibitors of the series. In contrast, substitution with the unsubstituted benzylthio moiety (IC₅₀?=?3.03?µM) resulted in significantly weaker inhibition activity towards MAO-B. This study suggests that quinazolinones are promising leads for the development of selective MAO-B inhibitors which may be used for the treatment of neurodegenerative disorders such as Parkinson’s disease.     

Comparative study of inhibitory specificity of liver monoamine oxidase in frogs <Emphasis Type="Italic">Rana ridibunda</Emphasis> and <Emphasis Type="Italic">Rana temporaria</Emphasis>

A comparative enzymological investigation of inhibitory specificity of the liver monoamine oxidases (MAO) from the two frog species, lake frog Rana ridibunda and grass frog Rana temporaria, revealed certain interspecies similarities and distinctions of this enzyme. The anti-monoamine oxidase effect of five derivatives of acridine, three derivatives of phenothiazine and one derivative of xanthene (pyronine G) was comparatively analyzed. The tested six-membered tricyclic compounds were shown to exert an irreversible inhibitory effect on the enzyme from both biological sources, displaying the same substrate deamination specificity. Thus, the rate of interaction of acridine and phenothiazine derivatives with the MAO active center in both frog species was considerably higher when activity was determined using noradrenaline versus N-methylhistamine, while that of pyronine G—when activity was determined using N-methylhistamine versus noradrenaline. Interspecies quantitative differences were found in the inhibitory efficacy and degree of selectivity of the tested tricyclic compounds, indicative of the differences in catalytic properties of liver MAO at the interspecies level in the representatives of the genus Rana, family Ranidae. The data of substratespecific inhibitory analysis provide indirect evidence of the existence of two molecular MAO forms in the liver of the studied frog species.