Amine oxidase histochemistry of the human uterus during the menstrual cycle

Summary The enzymes monoamine oxidase A (MAO A), monoamine oxidase B (MAO B) and benzylamine oxidase (BzAO) have been localized histochemically in the human uterus during various phases of the menstrual cycle. The results show a large increase in MAO A activity in the endometrial gland cells in the secretory phase of the cycle. MAO B activity was found in both endometrium and myometrium but did not show a cyclical variation in activity. BzAO was localized primarily in the tunica media of the myometrial blood vessels. These observations have been supported by parallel biochemical assays.This research was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CN Pq), Brazil and The Wellcome Trust, U.K., who defrayed the salary of Rachel Lewinsohn     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

Increased monoamine oxidase and semicarbazide-sensitive amine oxidase activities in white adipose tissue of obese dogs fed a high-fat diet

Adipocytes express two types of amine oxidases: the cell surface semicarbazide-sensitive amine oxidase (SSAO) and the mitochondrial monoamine oxidase (MAO). In human abdominal subcutaneous adipose tissue, it has been reported that SSAO substrates stimulate glucose transport and inhibit lipolysis while MAO activity is decreased in obese patients when compared to age-matched controls. However, no information has been reported on visceral WAT. To further investigate the obesity-induced regulations of MAO and SSAO in white adipose tissue (WAT) from different anatomical locations, enzyme activities and mRNA abundance have been determined on tissue biopsies from control and high-fat fed dogs, an obesity model already described to be associated with arterial hypertension and hyperinsulinemia. MAO activity was increased in the enlarged omental WAT of diet-induced obese dogs, but not in their mesenteric WAT, another intra-abdominal fat depot. Subcutaneous WAT did not exhibit any change in MAO activity, as did the richest MAO-containing tissue: liver. Similarly, SSAO was increased in omental WAT of diet-induced obese dogs, but was not modified in other WAT and in aorta. The increase in SSAO activity observed in omental WAT likely results from an increased expression of the AOC3 gene since mRNA abundance and maximal benzylamine oxidation velocity were increased. Finally, plasma SSAO was decreased in obese dogs. Although the observed regulations differ from those found in subcutaneous WAT of obese patients, this canine model shows a tissue- and site-specific regulation of peripheral MAO and SSAO in obesity.     

Localization of rat striatal monoamine oxidase activities towards dopamine,serotonin and kynuramine by gradient centrifugation and nigro-striatal lesions

Subfractionation of the crude synaptosomal-mitochondrial fraction of rat striatum in a continuous sucrose gradient in a zonal rotor led to the following results. The distribution pattern of monoamine oxidase (MAO) activity towards dopamine (DA) was very similar to the pattern of MAO activity towards serotonin (5HT), but differed from the pattern of MAO activity towards kynuramine (KYN). As 5HT is specifically deaminated by MAO-A while KYN is a common MAO substrate, this supports earlier suggestions that in rat striatal preparations DA is deaminated preferentially by MAO-A. The patterns of the MAO activities towards DA and 5HT were clearly dissimilar, despite considerable overlap, to the patterns of tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) activity, both marking the presence of striatal dopaminergic synaptosomes. The peak activities were separated and all patterns were symmetrical without showing a shoulder. This indicates that rat striatal MAO activity towards DA and 5HT is not specifically or for the greater part localized in dopaminergic terminals. We also investigated the effects of electrolytic and 6-hydroxydopamine lesions of the substantia nigra, both causing extensive degeneration of striatal dopaminergic terminals as appeared from the large decrease of striatal TH and DD activity. However, neither type of lesion induced a reduction of the MAO activity towards any of the substrates used. It is concluded towards DA and 5HT (probably MAO-A activity) present in dopaminergic terminals is very low compared with the total activity of this enzyme in rat striatal tissue.     

Effects of tobacco smoke constituents on MPTP-induced toxicity and monoamine oxidase activity in the mouse brain

Exposure to cigarette smoke has been found to attenuate the reduction in striatal dopamine levels caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice and to inhibit monoamine oxidase (MAO) activity in brain tissue. To confirm whether specific smoke constituents which have been reported to protect against MPTP toxicity were responsible for these effects, mice were treated chronically with nicotine, 4-phenylpyridine and hydrazine. Although all three compounds prevented the decrease in dopamine metabolite levels induced by MPTP, there was no significant effect on dopamine levels. None of the three compounds inhibited MAO activity in cerebral tissue following treatment in vivo. However, an extract of tobacco smoke particulate matter caused a marked inhibition of MAO A and MAO B activity when added in vitro. The results suggest that one or more unidentified substances in tobacco smoke are capable of inhibiting brain MAO and perhaps altering the formation of the active metabolite of MPTP.     

Cord blood amine oxidase activities relate to arousal and motor functioning in human newborns

To examine relationships between amine oxidase activities and behavior prior to significant postnatal experience, umbilical cord blood samples were collected from 28 normal infants, and the Brazelton Neonatal Behavioral Assessment Scale was administered within 72 hours of birth. Infants with lower platelet monoamine oxidase (MAO) and/or lower plasma amine oxidase activity were more highly aroused, more motorically active and more difficult to console than those with higher MAO activity. These behavioral characteristics are remarkably similar to the more elaborate affective and social features which have previously been associated with MAO activity differences in adult humans and rhesus monkeys.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Localization of some steroidogenic enzymes in the ovary of the rabbit

The distribution of delta 5-3 beta-HSD, peroxidase and cytochrome oxidase in immature, sexually mature and pregnant rabbit ovary has been studied histochemically. Corpora lutea are found only in pregnant rabbits. delta 5-3 beta-HSD is present in the theca interna of mature follicles, corpora lutea and interstitial gland cells but is absent in the granulosa cells of both developing and mature follicles. The granulosa cells of mature and developing follicles, hypertrophied theca interna and the luteal cells all show intense cytochrome oxidase activity. Peroxidase is present in the corpora lutea only. It is suggested that delta 5-3 beta-HSD in the theca interna and interstitial gland cells is the enzyme responsible for steroid synthesis in the ovaries of immature as well as sexually mature rabbits, while peroxidase and delta 5-3 beta-HSD present in the corpora lutea together regulate luteal steroidogenesis during pregnancy. The intense cytochrome oxidase activity together with peroxidase and delta 5-3 beta-HSD confirms the observations that this tissue is a site of intense oxidative activity.     

Biochemical characteristics of liver and brain monoamine oxidase from pacu

Monoamine oxidase (MAO) activity in the liver and brain of the pacu, Piaractus mesopotamicus was determined using a fluorescence assay with kynuramine as substrate. Apparent Michaelis constant values (20·33 μM for liver and 25·85 μM for brain) were similar in these tissues, but in terms of tissue protein MAO activity from liver was 4·5 times higher than from brain. The greater inhibitory effects of clorgyline than of deprenyl on MAO activity from liver and brain of this species suggest that pacu's MAO is a type A-like enzyme.     

Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A.

To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.     

Some characteristics of monoamine oxidase activity in tissues of the adult female grassfrog (Rana pipiens)

1. Monoamine oxidase (MAO) activity was measured fluorometrically in tissues of the grass-frog, Rana pipiens. 2. Incubation with specific inhibitors revealed the presence of two forms of MAO, type A and type B, in all tissues examined, though the ratios were different in each tissue. 3. Liver contained the greatest, and oviduct and skeletal muscle the lowest amounts of MAO activity, and brain also contained relatively high MAO levels. 4. The apparent Michaelis constant (Km) was similar in most tissues but was higher in pancreas, muscle and oviduct. Values of Km were lower than those reported for mammalian tissues.     

Peroxidase and Polyphenol Oxidase Associated with, Induced Resistance of Mung Bean to Rhizoctonia solani Kuhn

Peroxidase and polyphenol oxidase activities and peroxidase isozyme patterns were determined at different stages of hypocotyls of mung bean infected with Rhizoctonia solani. The effect of ethephon (2-chloroethyl phosphonic acid) has been studied. Peroxidase activity increased at 24 h after inoculation as compared with controls followed by a decline later. It increased at 120 h. Polyphenol oxidase activities increased after inoculation. Ethephon treatment increased the resistance to Rhizoctonia solani and enhanced peroxidase and polyphenol oxidase activities. Peroxidase isozyme pattern was found to change as a result of inoculation and ethephon treatment. The results indicated that ethephon-induced resistance was related to peroxidase and polyphenol oxidase activities.     

Cardiac levels of fibronectin,laminin, isomyosins,and cytochrome c oxidase of weanling rats are more vulnerable to copper deficiency than those of postweanling rats

The relative quantities of cardiac laminin, fibronectin, cytochrome c oxidase (CCO), and isomyosin types were studied by gel electrophoresis in male rats fed copper-deficient diets beginning either from the time of weaning for 5 weeks or from 5 weeks postweaning for 6 weeks with one group of copper-repleted rats. Increased levels of fibronectin and V(3) isomyosin but decreased levels of CCO subunit IV and laminin were found in weanling copper-depleted rats. In contrast, postweanling copper-depleted rats exhibited only increased levels of fibronectin and decreased levels of cardiac CCO subunit IV. Repletion of copper-deficient rats for 6 weeks was not sufficient to restore CCO subunit IV to the same level as controls. These results confirm that biochemical lesions in the basal laminae are a result of copper restriction. The decreased nuclear encoded subunits of CCO may help explain some of the mitochondrial pathology observed in dietary copper restriction. Increased V(3) isomyosin levels with low ATPase activity may help to conserve to a limited extent the ATP levels in copper-deficient cardiac tissue. These protein changes are consistent with the known morphological alterations of hearts from copper-restricted rats.     

Influence of high-fat diet on amine oxidase activity in white adipose tissue of mice prone or resistant to diet-induced obesity

Decreased monoamine oxidase (MAO) activity has been observed in adipose tissue of obese patients. Since substrates of MAO and semicarbazide-sensitive amine oxidase (SSAO) can modify adipocyte metabolism, this work investigates whether changes in amine oxidase activity may occur during white adipose tissue (WAT) development. We evaluated MAO and SSAO activities in WAT of high-fat diet (HFD) and low-fat diet fed mice. To distinguish the effect of HFD on its own from the effect of fat mass enlargement, obesity-prone transgenic line of the FVBn strain lacking beta3-adrenergic receptors (AR) but expressing human beta3-AR and alpha2-AR (mbeta3-/-, hbeta3+/+, halpha2+/-) was compared to its obesity-resistant control (mbeta3-/-, hbeta3+/+). As already reported, the former mice became obese while the latter resisted to HFD. No significant change in SSAO or MAO activity was found in WAT of both strains after HFD when expressing oxidase activity per milligram of protein. However, when considering the overall capacity of the fat depots to oxidize tyramine or benzylamine, there was an increase in MAO and SSAO activity only in the enlarged WAT of HFD-induced obese mice. Therefore, the comparison of these models allowed to demonstrate that the higher amine oxidase capacity hold in enlarged fat stores of obese mice is more likely the consequence of increased fat cell number rather than the result of an increased expression of MAO or SSAO in the adipocyte.     

Evidence for a single catalytic binding site on human brain type B monoamine oxidase

The tricyclic antidepressant drug, amitriptyline, inhibited the B form of human brain mitochondrial monoamine oxidase (MAO) under normal atmospheric conditions in a noncompetitive manner when phenylethylamine (PEA) was used as substrate and competitively when benzylamine (BzNH2) was employed as substrate. In addition, it was also found that PEA and BzNH2 inhibited each other's degradation noncompetitively. Similar results have previously been reported with human platelet MAO. These data suggest that the catalytic binding sites for PEA and BzNH2 on the B form of human brain MAO may be different. Attempts were made to further distinguish these catalytic binding sites on the brain oxidase using the irreversible MAO inhibitors, pargyline and clorgyline. Though these drugs have considerably different affinities for the B form of the oxidase, the degree to which either compound inhibited PEA or BzNH2 deamination was essentially identical. When incubations were performed at elevated oxygen concentrations PEA and BzNH2 became mutually competitive inhibitors of each other's metabolism. Also at the higher levels of oxygen, amitriptyline inhibition of PEA deamination approached a competitive fashion. These results suggest that PEA and BzNH2 share a common catalytic binding site on the B form of MAO and, in addition, bind to an inhibitory site on the reduced form of the oxidase. Accordingly, the data indicate that amitriptyline also binds to both the oxidized and reduced forms of this human brain oxidase.     

Influence of prolonged fasting on monoamine oxidase and semicarbazide-sensitive amine oxidase activities in rat white adipose tissue

Monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) activities are very high in white adipose tissue (WAT). SSAO, also known as Vascular Adhesion Protein-1 in vessels, is present at the surface of fat cells and independent approaches have evidenced its impressive increase during adipogenesis. However, the factors that might regulate the expression SSAO and MAO in adipose tissue are still poorly defined. Here, we report the influence of fasting on MAO and SSAO activities in adipose depots. A decrease of MAO activity occurred after three days of starvation in the intra-abdominal adipose tissue (INWAT) of male Wistar rats, regardless of their initial adiposity or fat loss. The reduced fat stores of seven-week old rats, loosing 59% of INWAT mass during fasting, contained only one half of the MAO activity found in fed control. The same reduction of MAO was observed after prolonged fasting in older rats which lose only 26% of their INWAT during the same starvation duration, leading to a fat mass comparable to that of younger fed control rats. It was therefore the endocrine and metabolic changes occurring during fasting that were responsible for the reduced MAO activity and not the amount of INWAT. Surprisingly, SSAO activity remained unchanged during starvation. In subcutaneous WAT, the changes in MAO and SSAO activities exhibited the same tendencies than those found in INWAT. Taken together, these data show that both MAO and SSAO activities increase in INWAT with age-dependent fattening, and indicate that only MAO diminishes during fasting.     

Irreversible inhibition of monoamine oxidase by some components of cigarette smoke

Inhibitory activity towards monoamine oxidase has been found in a solution of cigarette smoke. The inhibition was irreversible. When tissue slices of rat lung were incubated in the cigarette smoke solution or alternatively, exposed directly to cigarette smoke, monoamine oxidase activities were reduced drastically. Similarly, human saliva after cigarette smoking also exhibits considerable MAO inhibitory activity. When the amine substrates p-tyramine, serotonin and beta-phenylethylamine were incubated with the cigarette smoke solution, lipophilic adducts were formed non-enzymatically. The irreversible inhibition of MAO by cigarette smoke may well be related to the low platelet MAO associated with cigarette smokers as previously reported. The implication of such cigarette smoke-caused reduction of MAO activity in relation to Parkinsonism is discussed.     

Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.     

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.     

Contribution of monoamine oxidase (MAO) inhibition to tobacco and alcohol addiction

Whole-body PET-scan studies in brains of tobacco smokers have shown a decrease in monoamine oxidase (MAO) activity, which reverts to control level when they quit smoking. The observed decrease in MAO activity in smokers is presumably due to their exposure to tobacco constituents that possess MAO-inhibiting properties. The inhibition of MAO activity seems, however, not to be a unique feature of tobacco smoking as subjects with Type II alcoholism have been reported to show a similar decrease in MAO activity that reverses when they cease to use alcohol. The present review summarizes the data on MAO-inhibiting tobacco constituents and explains that the decrease in MAO activity observed in alcoholics is probably due to concomitant tobacco use. It is concluded that the inhibition of MAO by constituents contained in tobacco and tobacco smoke, enhances the addiction induced by tobacco smoking.