Species differences in lung mitochondrial monoamine oxidase activities

Pulmonary mitochondrial monoamine oxidase (MAO) activity was examined in preparations from rat, rabbit and guinea-pig with 12 different amines as substrates: serotonin, norepinephrine, and octopamine (type A specific); tryptamine, benzylamine, 5-methoxytryptamine, 5-methyltryptamine, p-methoxyphenylethylamine, and 3,4-dimethoxyphenethylamine (type B specific); and tyramine, dopamine and 3-methoxytyramine (type A + B specific). The oxidation of type A and type A + B substrates was greater in guinea-pig lung mitochondria than in rat or rabbit preparations. Except for benzylamine, the oxidation of type B substrates was similar in all three species. Benzylamine was not oxidized by guinea-pig lung mitochondria but was actively metabolized by rat and rabbit preparations.     

Possible mechanism of selective inhibition of rat liver mitochondrial monoamine oxidase by chlorgiline and deprenyl]

The inhibition of the deamination of serotonin (the main substrate of monoamine oxidase (MAO) type A) by chlorgiline and deprenyl and of beta-phenylethylamine (the main substrate of the B type MAO) by fragments of rat liver mitochondrial membrane as well as the influence of 4-ethylpyridine on this process were studied. It was shown that the MAO activity of the mitochondrial membrane fragments was highly sensitive to chlorgiline, when serotonin was used as substrate, whereas a high sensitivity toward deprenyl was observed with beta-phenylethylamine as substrate. 4-Ethylpyridine (5.10(-3) M), a competitive and reversible inhibitor of the MAO activity, inhibited deamination of serotonin and beta-phenylethylamine by 34 and 30%, respectively. In experiments with chlorgiline (the specific inhibitor of MAO type A) 4-ethylpyridine (5.10(-3) M) introduced into the samples after preincubation of mitochondria with increasing concentrations of chlorgiline (30 min, 23 degrees C) decreased the inhibition by chlorgiline of the deamination of beta-phenylethylamine, but sharply increased the inhibitory effect of chlorgiline on the oxidation of serotonin. In analogous experiments with deprenyl (the specific inhibitor of MAO type B) 4-ethylpyridine (5.10(-3) M) decreased the inhibitory effect of deprenyl not only on the deamination of serotonin (substrate of MAO A), but also on the oxidation of beta-phenylethylamine (the main substrate of MAO type B). The decrease in the inhibitory effect of deprenyl on the deamination of beta-phenylethylamine after the addition of 4-ethylpyridine may be intensified upon preincubation of deprenyl with mitochondria in the presence of 4-ethylpyridine. The data obtained demonstrate the difference in the type and mechanism of inhibition of the deamination of serotonin by chlorgiline as well as in the type and mechanism of oxidation of beta-phenylethylamine by deprenyl. The possible mechanism of selective blocking of MAO activity by chlorgiline and deprenyl was discussed in terms of our previous data on the existence in the active center of mitochondrial MAO of specific sites for substrate binding, differing in their structure-functional characteristics.     

Enhanced selectivity of pargyline as an inhibitor of type B monoamine oxidase in harmaline-treated rats

Pargyline, a slightly selective inhibitor of type B monoamine oxidase (MAO), inhibited phenylethylamine oxidation by 88 ± 1% and 81 ± 1% in rat brain and liver, respectively, at 24 hrs after injection of a 30 mg/kg i.p. dose. Serotonin oxidation was inhibited to a lesser extent, 68 ± 4% and 68 ± 2%, respectively, in brain and liver. In rats treated with harmaline, a short-lasting reversible MAO inhibitor selective for type A MAO, the inhibition of phenylethylamine oxidation after pargyline injection still occurred but the inhibition of serotonin oxidation was prevented. These results illustrate that a selective MAO inhibitor can be used to enhance the selectivity of an irreversible inhibitor, presumably by occupying active sites on a certain form of MAO temporarily and thereby preventing its inactivation. In heart, inhibition of both phenylethylamine and serotonin oxidation by pargyline was prevented by harmaline; this finding supports other evidence that phenylethylamine is metabolized by type A MAO in rat heart.     

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.     

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.     

The effect of isatin (tribulin) on metabolism of indoles in the rat brain and pineal: In vitro and in vivo studies

Isatin (Tribulin) produced a dose-dependent inhibition of both MAO A and MAO B in broken cell preparations from rat brain and pineal. However, isatin administered in vivo (80–160 mg/kg) to the intact animal significantly increased brain, but not pineal, serotonin and did not affect 5HIAA or other indoles in either brain or pineal. Further, in vivo administration did not produce detectable MAO inhibition in either tissue. In pineal organ culture, addition of isatin up to 1mM had no influence on the concentrations of pineal indoles or the activities of monoamine oxidase or serotonin N-acetyltransferase. However, the diazepam augmentation of beta adrenergic induction of serotonin N-acetyltransferase activity was blocked by isatin. The results of these studies call into question the proposed role of isatin as an endogenous monoamine oxidase inhibitor but support a possible role as a benzodiazepine receptor blocker.     

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.     

Inhibition of rabbit monoamine oxidase by doxepin and related drugs.

The psychotherapeutic agent, doxepin, inhibits both the B and A forms of rabbit lung mitochondrial monoamine oxidase (MAO). The K_i values for doxepin inhibition of phenylethylamine (PEA) and 5-hydroxytryptamine (5-HT) deamination is 3 × 10⁻⁵ and 2 × 10⁻⁴M, respectively. Doxepin is, thus, similar to other tricyclic antidepressant drugs in that it has a greater affinity for the B form of the rabbit oxidase. The ability of doxepin and imipramine and its mono and didesmethyl derivatives to inhibit rabbit MAO is decreased as the pH is raised above 8.0. However, results suggest that the basicity of the propylamine side chain has little or no influence on the ability of these drugs to inhibit the rabbit oxidase. In addition, it is demonstrated that 7 × 10⁻⁶M doxepin inhibits PEA deamination by human platelet MAO approximately 50%.     

Stress-induced synthesis of melatonin: possible involvement of the endogenous monoamine oxidase inhibitor (tribulin)

Cold-restrained stress increased rat pineal melatonin and N-acetylserotonin content. This effect was partially prevented by lorazepam. Serotonergic turnover (ratio of 5-hydroxyindole acetic acid to serotonin) was significantly decreased in stressed but not in stressed rats pretreated with lorazepam, suggesting stress-induced inhibition of monoamine oxidase (MAO). Literature data indicate that the same type of stress increases the production of the endogenous MAO inhibitor. The implication of stress-induced MAO inhibition on melatonin synthesis in anxiety and drug withdrawal is discussed.     

Interaction of N-(2-Nitro-4-Azidophenyl)-Serotonin with Two Types of Monoamine Oxidase in Rat Brain

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.     

Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

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 activity of rat brain under cold exposure

Cold stress and cold adaptation were studied for their effect on the activity and substrate specificity of the monoamine oxidase A and B and on the Km of serotonin deamination in the rat brain mitochondria and supernatant. Mitochondrial monoamine oxidase Km with serotonin is established to increase more than twice under cold stress and decrease considerably in cold adapted rats. The lowering of the mitochondrial monoamine oxidase A activity is accompanied by the appearance of serotonin and the glucosamine deaminating activity in supernatant. The data suggest that decrease in the monoamine oxidase activity under cold stress may be caused by both release of the enzyme from mitochondrial membrane and changes in its catalytic property alteration.     

Alteration of dopamine synthesis in rat striatum subsequent to selective type A monoamine oxidase inhibition

Abstract: Pretreatment of rat striatal slices with the selective type A monoamine oxidase (MAO) inhibitor clorgyline was found to produce significant inhibition of dopamine (DA) synthesis. DA synthesis was reduced by nearly 50%, but not until more than 90% of the type A enzyme was inhibited. In contrast, complete inhibition of the type B MAO following deprenyl treatment had no effect. It is suggested that interneuronal accumulation of DA following inhibition of type A MAO leads to feedback inhibition at the rate-limiting step in DA biosynthesis, tyrosine hydroxylation. These results are also consistent with the presence of a type A MAO within DA-containing neurons and provide evidence of a regulatory role for type A MAO in the synthesis of brain DA.     

Methyl parathion induced alterations in monoaminergic system of developing rat pups

Methyl parathion induced alterations in the level of monoamines, viz. norepinephrine, dopamine and serotonin were studied in discrete regions of developing central nervous system of rat pups. A significant decrease in the level of monoamines noticed in methyl parathion toxicosis may be related to the altered neuronal activity and inefficiency, leading to depression and impairment in various behavioural activities. In contrast to AChE inhibition, monoamine oxidase (MAO) activity showed an increasing trend and it could cause deamination of catecholamines and accumulation of its metabolites. This suggests that an increased AChE inhibition may indirectly stimulate MAO activity in developing rat pups exposed to methyl parathion.     

Comparative Study of Catalytic Properties of Monoamine Oxidases of the Liver of the Squid Todarodes pacificus and of the Liver of the Wistar Rat

A comparative study of substrate specificity of monoamine oxidase (MAO) in mitochondria of liver of the Pacific squid Todarodes pacificus and of Wistar rats is carried out. It is revealed that the squid liver MAO, unlike the rat liver MAO, is capable of deaminating not only tyramine, serotonin, and benzylamine, but also histamine. The squid liver MAO activity in relation to all studied substrates is approximately 10 times lower, while the sorption ability, several tens times lower, than the rat liver MAO. Semicarbazide, a classic inhibitor of diamine oxidase, at a concentration 1 × 10^–2 M did not inhibit the catalytic activity of both studied enzymes. The specificity of action of an irreversible inhibitor, proflavine, is established, which was seen at deamination of various substrates by the squid liver MAO to the greater degree, than by the rat liver MAO. The values of the bimolecular rate constant of the irreversible inhibition (k _II) by proflavine were 2.5–20-fold higher (depending on substrate) in the case of the squid liver MAO, than of the rat liver MAO. A suggestion is put forward about the probable presence of several centers of substrate binding in the enzyme of the studied marine invertebrate, like in the mammalian enzyme.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.     

Neuromediator mechanisms of the effect of the antihistamine agent dimebone on the brain

Dimebone was shown to inhibit monoamine oxidase (MAO) deaminating dopamine and serotonin, decrease dopamine metabolism in the basal ganglia of the rat brain, increase noradrenaline level and depress dopamine deamination in the hypothalamus. Dimebone first increased and then diminished the release of dopamine in the cortex, with the concomitant MAO activation and the increase in dopamine and noradrenaline levels. The in vitro experiments have demonstrated that dimebone (10(-4)) preferentially inhibited MAO activity, type B and dopamine deamination in homogenates of different rat brain structures. The role of MAO inhibition in the mechanism of dimebone action on the catecholamine metabolism in the brain structures and its stimulating effect on CNS are discussed.