Studies of monoamine oxidases: Inhibition of bovine brain MAO in intact mitochondria by selective inhibitors

The inhibition of mitochondrial monoamine oxidase (MAO) from beef brain cortex by the selective inhibitors, clorgyline, harmaline, Deprenyl and pargyline, was compared using five substrates: serotonin (5-HT), β-phenylethylamine (PEA), tyramine, tryptamine and dopamine. Dose-response studies, consistent with the classification of MAO, types A and B, indicated that serotonin deamination was more sensitive to clorgyline and harmaline inhibition than was phenylethylamine. However, the curves for all substrates were double-sigmoidal, rather than being a single sigmoid curve for 5-HT and PEA. Deprenyl and pargyline did not exhibit any marked selectivity for inhibiting PEA deamination without prior preincubation of enzyme and inhibitor. The rate of inhibition was variable and was dependent upon the substrate, the nature of the inhibitor and the inhibitor concentration. Dual inhibitor studies, using the “type A” inhibitor, clorgyline, and the “type B” inhibitor, Deprenyl, together, resulted in almost complete MAO inhibition, regardless of substrate. Combining the two type A inhibitors, clorgyline and harmaline, or the two type B inhibitors, deprenyl and pargyline, resulted in inhibitions that were equal to or only slightly greater than the inhibition produced by a single inhibitor. These results suggested that there are at least two distinct sites in beef brain MAO from cortical mitochondria which may be interacting. The deamination of all substrates occurs at both sites.     

Effects of tricyclic antidepressant drugs on energy-linked reactions in mitochondria

The effects of impramine and chlorimipramine on energy-linked reactions in mitochondria were characterized. Both compounds exhibited some characteristics of classical uncouplers of oxidative phosphorylation, i.e. they released respiratory control, hindered ATP synthesis, and enhanced ATPase activity of isolated rat liver mitochondria. Unlike classical uncouplers, however, these compounds only weakly stimulated proton uptake in intact mitochondria. They also exhibited unusual effects on energy-linked reactions in beef heart submitochondrial particles (SMP). Both compounds inhibited NADH oxidation in SMP in an "oligomycin-like" manner, and inhibited ATPase activity of SMP and the soluble F1-ATPase. In contrast, the drugs weakly inhibited ATPase activities of bovine adrenal gland chromaffin granules and resealed granule ghosts. The mechanisms responsible for the multiple effects on mitochondrial energy-linked processes are unclear. They may be related to the hydrophobicity of the drugs, as has been shown for other hydrophobic amines.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Inhibition of brain mitochondrial monoamine oxidases by the endogenous compound 5-hydroxyoxindole

5-Hydroxyoxindole is a recently identified endogenous compound. Its physiological role remains unclear but certain evidence exists, that it may share some regulatory properties with isatin, a known endogenous inhibitor of monoamine oxidase (MAO) type B (MAO-B). In this study several oxidized indoles were tested for their in vitro inhibition of MAO type A (MAO-A) and B of rat brain non-synaptic mitochondria. 5-Hydroxyoxindole was less potent MAO-A inhibitor (IC50 56.8 microM) than isatin (31.8 microM) and especially 5-hydroxyisatin (6.5 microM), but it was the only highly selective MAO-A inhibitor among the all compounds studied (IC50 MAO-A:IC50 MAO-B = 0.044). Thus, the in vitro data suggest that MAO-A may represent potential target for 5-hydroxyoxindole.     

Effect of antidepressant drugs on monoamine synthesis in brain in vivo.

The activity of tryptophan and tyrosine hydroxylase were estimated in vivo by measuring the accumulation during 30 min of 5-hydroxytryptophan (5-HTP) and 3,4-dihydroxyphenylalanine (DOPA), respectively, after inhibition of aromatic amino acid decarboxylase by administration of m-hydroxybenzylhydrazine (NSD 1015) (100 mg/kg, i.p.). Whereas the activity of tyrosine hydroxylase in the dopamine-rich striatum was sensitive to haloperidol, which caused a significant increase in accumulation of DOPA, there was no effect of haloperidol in the predominantly noradrenergic frontoparietal cortex, confirming that the activity of tyrosine hydroxylase, measured in the frontoparietal cortex, is essentially localized in noradrenergic neurones. In the frontoparietal cortex of the rat the in vivo activity of tryptophan and tyrosine hydroxylase were equipotently attenuated by imipramine, while the selective blocker of the uptake of noradrenaline, desipramine and the selective blocker of the uptake of serotonin, citalopram, reduced only tyrosine or tyrosine hydroxylase respectively. Milnacipran, an antidepressant which inhibits the uptake of both monoamines to a similar extent, decreased the synthesis of both monoamines equipotently. The monoamine oxidase inhibitor, clorgyline, also reduced the synthesis of both monoamines. Thus, the in vivo inhibition of the synthesis of monoamines would appear to be mediated by an increase in synaptic concentration of monoamines, resulting from the inhibition of the uptake or catabolism of monoamines. Chronic administration of citalopram led to a significant increase of the basal synthesis of 5-hydroxytryptamine (5-HT). Milnacipran, given chronically, significantly enhanced the basal synthesis of both 5-HT and noradrenaline (NA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of tricyclic antidepressant drugs with human and rat monoamine oxidase type B

Summary The effect of tricyclic antidepressant drugs on the deamination of phenylethylamine and benzylamine by monoamine oxidase (MAO) type B was investigated in vitro in human brain cortex, human platelet, and rat brain preparations. These drugs inhibited MAO activity as expected; however, an atypical biphasic response was observed with the tertiary amine tricyclic, clomipramine, and, to a somewhat lesser extent, with two other tertiary amine tricyclics, imipramine and amitriptyline, when benzylamine was used as the substrate in human tissue preparations. This atypical biphasic pattern was not found when we used the secondary amine antidepressant drugs, desipramine, desmethylclomipramine, or fluoxetine, or used phenylethylamine as the substrate, or used rat rather than human brain tissue. For the tricyclics exhibiting normal inhibition patterns, the same rank order of inhibition was observed with benzylamine as a substrate in all three types of tissue; however with phenylethylamine, differences in inhibition were found between rat and human tissues. These tricyclic-MAO interactional data suggest that secondary and tertiary amine tricyclics interact differently with human MAO type B, that rat and human MAO type B are not functionally identical, and also support other data that phenylethylamine and benzylamine are deaminated by different mechanisms. Send offprint requests to A. A. Reid at the above address     

Reactive oxygen species production by monoamine oxidases in intact cells

Monoamine oxidase (MAO) A and B are mitochondrial enzymes involved in the oxidative deamination of endogenous and exogenous amines. At present, the production of H₂O₂ by MAO in intact cells and its functional consequences in cell function have not been extensively investigated. The aim of this study was to define whether, in intact cells, the metabolism of small amounts of MAO substrates was able to induce a detectable H₂O₂ production. Hydrogen peroxide production was measured using a luminol-amplified chemiluminescence assay in three cell types, rat mesangial cells, rabbit proximal tubule cells and Hep-G2 cells, containing different MAO A/MAO B ratios. Our results showed that cell incubation with tyramine (50 μmol/l) led to a time-dependent H₂O₂ generation which was fully inhibited by MAO A (clorgyline and RO 41–1049) and MAO B (selegiline and RO 19–6327) inhibitors. The extent of inhibition of H₂O₂ production by selective inhibitors was in agreement with the amount of MAO isoforms expressed in each cell type, as determined by Western blot analysis and enzyme assay. Altogether, these findings show that, in a normal cell environment, MAO can be a source of reactive oxygen species which could have a functional impact on cell functions. In addition, we propose the luminol-amplified chemiluminescence assay as a rapid and sensitive procedure to characterize the monoamine oxidase isoforms and their regulation in intact cells. Received: 15 December 1998 / Accepted: 17 February 1999     

Molecular structure and dynamics of tricyclic antidepressant drugs.

The molecular structure, electrostatic potentials and dynamics of imipramine, chlorimipramine, amitriptyline and nortriptyline were examined by computer graphics, molecular mechanical energy calculations and molecular dynamics simulations, using the AMBER all atom force field. Starting coordinates for amitriptyline and nortriptyline were generated by model building from the crystal structure of imipramine. The structures were refined by molecular mechanical energy minimization, and used as starting points for molecular dynamics simulations in vacuo and in aqueous solution. The simulations demonstrated considerable flexibility of the molecules, both in the side chain and in the ring system, where the angle between the phenyl rings varied between 90 degrees and 168 degrees. The most frequently observed conformations of imipramine, chlorimipramine and nortriptyline during the simulations had the side chain folded above one of the phenyl rings, while amitriptyline showed both folded and extended side chain conformations during the simulations. The results may provide increased understanding of the molecular recognition and specificity of tricyclic antidepressant drugs in interaction with neurotransmitter receptor molecules.     

Effects of long-term treatment with dicyclic,tricyclic, tetracyclic,and noncyclic antidepressant drugs on monoamine oxidase activity in mouse brain

• 1.1. The individual long-term effects of the antidepressant drugs zimeldine, viloxazine, imipramine, amitriptyline, nortriptyline, maprotiline, or nomifensine, on brain mitochondrial monoamine oxidase (MAO) activity, were studied in mice that were given daily intraperitoneal injections (30 mg/kg) of these reagents for 4 weeks.
• 2.2. Both the A-form (MAO-A) and B-form (MAO-B) of MAO were inhibited after long-term administration of all the drugs except nortriptyline (MAO-A was not affected) and maprotiline (neither MAO-A nor MAO-B were affected).
• 3.3. Kinetic analysis showed a significant decrease in V_max values, and an increase in K_m values for MAO-B during treatment.
• 4.4. All seven drugs are competitive inhibitors of MAO-A, noncompetitive inhibitors of MAO-B, and were more potent in vitro for MAO-B.
• 5.5. MAO-A was inhibited by the following drugs (in ascending order of potency): nortriptyline, amitriptyline, imipramine, maprotiline, zimeldine, nomifensine, and viloxazine.
• 6.6. MAO-B was inhibited by the following drugs (in ascending order of potency): nortriptyline, imipramine, maprotiline, amitriptyline, zimeldine, nomifensine, and viloxazine.

     

Inhibition by Zn(2+) of A-form monoamine oxidase in monkey brain mitochondria

The effects of ZnSO(4) on mitochondrial monoamine oxidase (MAO) activity in monkey brain were compared with those in rat and rabbit, in vitro. After preincubation at 25 degrees C for 20 min with 1 microM ZnSO(4), MAO-A activity in monkey brain was about 50% using serotonin (5-HT) as a substrate, and the inhibition was proportional to the concentration of ZnSO(4). However, ZnSO(4) had no effect on MAO-B activity in monkey brain using beta-phenylethylamine (beta-PEA) as a substrate. The inhibition by ZnSO(4) of MAO-A activity was competitive and reversible. CdSO(4) also inhibits MAO-A, but not MAO-B in monkey brain mitochondria. ZnSO(4) did not inhibit either MAO-A or MAO-B activity in rat and rabbit brain mitochondria. These results indicate that the inhibiting action of Zn(2+) differs depending on animal species. In monkey brain mitochondria, MAO-A was highly sensitive to Zn(2+) and MAO-B was less sensitive. These results also suggest that Zn(2+) may regulate the level of catecholamine content in monkey brain.     

Interactions of tricyclic antidepressant drugs with deprivation-induced fluid consumption by rats.

Six tricyclic antidepressant drugs, representing both secondary and tertiary amines and four different ring systems, were examined for their effects on deprivation-induced fluid consumption by rats in a standardized test system. All compounds reduced fluid consumption with a potency ranking of: desipramine ? amitriptyline ? doxepin ? nortriptyline ? imipramine ? protriptyline. In a similar test situation, pretreatment with PCPA markedly reduced fluid consumption at 24 hr; fluid consumption levels had not returned to normal by 15 days after dosage.     

Inhibition of monoamine oxidation in rat liver mitochondria.

The effects of various agents on inhibition of monoamine oxidase (MAO) and the amine dehydrogenating system (MADH) of rat liver mitochondria were studied. Cyanide and semicarbazide strongly inhibited MADH activity while MAO activity was not affected. MAO was found to be more susceptible to inhibition by tranylcypromine, pheniprazine, iproniazid and pargyline. The psychoactive agents tested inhibited MAO strongly whereas they inhibited MADH either feebly or not at all. Simultaneous addition of inhibitor and substrate did not show any appreciable inhibition of these enzyme systems by tranylcypromine, pheniprazine, iproniazid and pargyline. Degree of inhibition by these inhibitors was found to increase with increasing time of preincubation.