Contribution of Monoamine Oxidase(MAO) to the Binding of Tertiary Basic Drugs in Isolated Perfused Rat Lung

The effect of tertiary basic drugs on mitochondrial MAO activity and the effect of MAO inhibitors (MAOIs) on basic drug accumulation in the isolated perfused rat lung were studied to clarify the role of MAO in drug binding to lung tissue. In the perfused lung preparation, the inhibition of MAO by basic drugs correlated well with their lipid solubilities and followed competitive kinetics. The inhibitory rank order (imipramine diphenhydramine > quinine > metoclopramide > procainamide) also correlated with their accumulation in the perfused lung. Moreover, MAOI treatment decreased the accumulation of basic drugs in the lung, and the potency of MAOIs to inhibit drug accumulation in the lung correlated with their MAO inhibitory activity. These results indicate that lung MAO has specific binding sites for basic drugs and may function as a drug reservoir.     

Binding of Basic Drugs to Rat Lung Mitochondria

The role of the mitochondria in the accumulation of basic amine drugs in the rat lung was studied. Drug binding to the mitochondria was rapid and reached maximum levels after 2.5 min of incubation. Lipophilic basic drugs accumulated in the mitochondria more than nonlipophilic basic drugs and non-basic drugs, and the accumulation was dose dependent. Schatchard plots revealed at least two independent sets of binding sites for basic drugs in the mitochondria. The binding was competitively inhibited by other basic drugs but not nonbasic drugs. The degree of inhibition by competing basic drugs was correlated with their lipid solubilities. These findings with isolated mitochondria agree with previous results obtained with the perfused lung preparation and indicate that the mitochondria play an important role in the accumulation of basic drugs in the lung.     

Lights and shadows on monoamine oxidase inhibition in neuroprotective pharmacological therapies

Playing a pivotal role in the metabolism of neurotransmitters in the central nervous system, the mitochondrial enzymes monoamine oxidases A and B (MAO A and B) have been for long studied as drug targets for neurodegenerative and neurological diseases. MAO inhibitors (MAOIs) are clinically used to treat Parkinson's disease and depression by blocking the degradation of neuroactive catecholamines and providing a symptomatic relief in the patients. More recent is the idea that the neuroprotective effect of MAOIs may result from the prevention of oxidative stress produced by the MAO reaction rather than being simply related to the inhibition of neurotransmitters degradation. Tranylcypromine and phenelzine are among the first developed MAOI drugs and have been used for years to treat depression. Their usage is now limited to cases of refractory depression because of their negative side effects, which are due to both the lack of MAO A/MAO B selectivity and the inhibition of other enzymes such as the drug-metabolizing cytochromes P450. Although the multi-target action of these MAOIs determines negative implications, the most newly developed compounds have improved properties not only for their specificity relatively to MAO A/MAO B selectivity but also because they function through multiple mechanisms that produce beneficial effects. In particular, safinamide, a MAO B selective inhibitor in clinical trials for Parkinson's disease, is neuroprotective by blocking the voltage-dependent Na+ and Ca2+ channels and the Ca2+-mediated glutamate release processes. Rasagiline is a drug used in combination with L-dopa in the treatment of parkinsonian patients and the metabolic products of its degradation exert neuroprotective effects. Moreover, rasagiline scaffold is used to design analogs by addition of pharmacophores that act on other neurological targets. This multi-target approach may prove successful in order to find new and more effective therapies for the complexity of neurodegenerative diseases.     

Subcellular Distribution of Basic Drugs Accumulated in the Isolated Perfused Lung

To clarify the mechanism by which basic drugs accumulate in the lung, the binding selectivity of drugs to different subcellular structures of the perfused rat lung was examined. Imipramine, quinine, and metoclopramide were used as examples of basic drugs. The accumulation of basic drugs was highest in the mitochondrial fraction. The drug accumulation in the lung mitochondrial fraction increased with increasing lipid solubility and was dose dependent. These results suggest the presence of specific binding sites for basic drugs in mitochondria and that mitochondria play an important role as a reservoir for basic drugs.     

Use of a monoclonal antibody for comparative studies of monoamine oxidase B in mitochondrial extracts of human brain and peripheral tissues

Monoamine oxidase B (MAO B; EC 1.4.3.4) activity in detergent extracts of mitochondria from autopsy brain (gray matter and medulla), liver, lung, and kidney from a single individual and from pooled, human platelets could be immunoprecipitated by a monoclonal anti-human platelet MAO B antibody (MAO-1C2) in combination with appropriate secondary reagents. MAO A activity, which was detected in brain, liver, lung, and kidney, was not immunoprecipitated under the same conditions. All MAO B-containing extracts, regardless of tissue source, inhibited immunoprecipitation of [3H]pargyline-labeled human platelet MAO, and the shapes of the inhibition curves were identical. The concentration of immunologically detectable MAO B protein in the extracts was estimated from immunoprecipitation competition data by reference to a standard curve relating observed inhibition of immunoprecipitation to the concentration of catalytically active platelet MAO added (estimated from [3H]pargyline binding data). MAO B protein concentrations measured by this radioimmunoassay were similar to concentrations of active MAO B as measured by pargyline binding. These results demonstrate that in the brain and peripheral tissues studied, molecules with MAO B activity share a unique antigenic determinant and similar catalytic efficiency. They also extend previous observations that MAO B molecules extracted from mitochondria bear an antigenic determinant which is not present on MAO A molecules. These results demonstrate the validity of a new competitive radioimmunoassay for active plus inactive MAO B concentration in human platelet extracts and extracts of mitochondria from human tissues. This radioimmunoassay should complement [3H]pargyline binding assays and enzyme activity assays in studies designed to clarify the mechanisms of genetic, disease, and treatment factors which lead to differences in MAO B function among individuals.     

A comparative study of some kinetic and molecular properties of microsomal and mitochondrial monoamine oxidase

This experimental work tries to characterize the monoamine oxidase of microsomal origin through its kinetic and molecular properties, and to establish a comparative study with the enzyme present in rat liver mitochondria. The temperature effect upon this catalytic activity was examined and similar behaviour of MAO A and MAO B between both cellular fractions was found. The study of the pH dependence of initial velocity showed similar results both in mitochondria and in microsomes. The FAD cofactor is covalently attached to the MAO of microsomal origin. The FAD containing subunits corresponding to MAO A and MAO B, previous binding of the enzyme with [3H]pargyline and posterior SDS electrophoresis and fluorography, showed molecular weights of 65,900 and 62,400, respectively, in both cellular fractions. The inhibition curves with clorgyline, deprenyl, semicarbazide and KCN, measuring the remaining activity towards 1 microM of benzylamine, indicated that in mitochondria 5% of the total activity is due to the presence of SSAO activity whereas in microsomes this activity represents about 20%. From all these results it appears that mitochondrial and microsomal MAO are related enzymes, although further structural studies are necessary to confirm their possible identity.     

Multiple monoamine oxidase activities in heterogeneous populations of mouse lung mitochondria.

Both monoamine oxidase (MAO) A and B activities were almost exclusively found associated with mitochondrial fractions in mouse lung, and these activities could be partially separated on linear sucrose gradients. The peak MAO B activity measured by the deamination of β-phenylethylamine (PEA) was consistently found in a population of mitochondria sedimenting in a denser region of the gradient than peak MAO A activity for 5-hydroxytryptamine (5-HT). Clorgyline strongly inhibited deamination of 5-HT across the mitochondrial fractions, while deamination of PEA remained high. Pargyline blocked PEA deamination, while considerable activity remained for 5-HT. These results provide evidence for the possible existence of heterogenous subpopulations of lung mitochondria differing in sedimentation behavior and containing monoamine oxidase with different substrate specificity and inhibitor sensitivity.     

Effects of intraperitoneal cadmium administration on mitochondrial enzymes in rat tissues

The effects of cadmium on some mitochondrial enzymes of kidney, testis and lung were investigated in male rats. Rats were injected intraperitoneally with 3 mg Cd/kg body weight and its effects were studied 24, 72 and 144 h later. Succinic dehydrogenase and cytochrome c oxidase activities were significantly inhibited in kidney, testis and lung mitochondria. Citrate synthase activity was inhibited in testis mitochondria at all time periods studied, whereas in kidney and lung an initial increase in activity was followed by inhibition at later time periods.     

Styrene inhibits monoamine oxidase A, but not monoamine oxidase B in monkey brain mitochondria

The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.     

Effects of local anesthetics on monoamine oxidase, and their membrane effects

The effects of various local anesthetics on rat brain and liver monoamine oxidase (MAO) and their antihemolytic and local anesthetic effects were studied. All local anesthetics tested at 1 x 10(-7) M to 1 x 10(-3) M inhibited MAO activity in rat liver mitochondria with 5-hydroxytryptamine (5-HT) as substrate. The order of potency was tetracaine>procaine>dibucaine>lidocaine>prilocaine. Tetracaine and procaine inhibited 5-HT oxidation much more than beta-phenylethylamine (PEA) oxidation. Dibucaine inhibited PEA oxidation as much as 5-HT oxidation. Inhibition of MAO by local anesthetics other than dibucaine was reversible. Tetracaine and procaine inhibited 5-HT oxidation competitively, whereas dibucaine inhibited it non-competitively. Antihemolytic effects were observed with dibucaine and tetracaine at concentrations of 6 x 10(-5) M and 1 x 10(-4), respectively. The order of surface anesthetic potencies was dibucaine>tetracaine>prilocaine>lidocaine>procaine. These results suggest that the inhibition of MAO activities by local anesthetics depends on both electrostatic and hydrophobic interactions between these drugs and enzyme-associated phospholipids or the hydrophobic regions of proteins.     

Deamination of beta-phenylethylamine by monoamine oxidase--inhibition by imipramine

The oxidative deamination of tyramine (Tyr), 5-hydroxytryptamine (5-HT), and β-phenylethylamine (PEA) by mitochondrial preparations of rabbit lung and brain was inhibited by imipramine. This tricyclic iminodibenzyl antidepressant drug was most effective in decreasing the deamination of PEA: at 1 × 10^?4M imipramine, deamination of PEA, Tyr and 5-HT was inhibited by approximately 70, 45 and 45 per cent, respectively, when either lung or brain mitochondrial monoamine oxidase (MAO) preparations were used. Imipramine-induced inhibition of MAO was shown to be of a mixed type based on Lineweaver-Burk plots, but was found to be completely reversible. The desmcthyl and didesmethyl derivatives of imipramine were equally as effective as the parent drug in inhibiting the deamination of PEA, whereas the N-oxide analog of imipramine was less effective as an inhibitor of this reaction. These results support the premise that the action of imipramine as a clinically effective antidepressive agent may be related to its inhibitory effect on the specific form of MAO which deaminates PEA.     

Inhibition of mitochondrial monoamine oxidase of the rat uterus and liver by clorglyine, pargyline and harmine.

The inhibition of mitochondrial monoamine oxidase (MAO) activity in rat uterus and liver by clorgyline, harmine and pargyline is reported. MAO activity is shown to be present in mitochondria of the rat uterus by rate-zonal centrifugation on a sucrose gradient. Each inhibitor was tested for its ability to inhibit the oxidation of tyramine (TYN). 5-hydroxytryptamine (5HT) and β-phenylethylamine (PEA). TYN deamination by uterine organelles was inhibited in two distinct steps by clorgyline and harmine, whereas in liver mitochondria only clorgyline manifested the two-step inhibition pattern. Elimination of TYN oxidation by pargyline occurred as a single sigmoid curve. Single sigmoid inhibition curves with all three inhibitors were also observed for 5HT and PEA in both tissues. For uterine and liver mitochondria the relative effectiveness of each inhibitor toward the oxidation of the three substrates was as follows: (a) clorgyline and harmine. 5HT > TYN > PEA; (b) pargyline. PEA > TYN > 5HT. It was concluded that, as has been previously demonstrated in liver, two forms of MAO exist in mitochondria isolated from the rat uterus. This conclusion is based upon (1) the biphasic inhibition of TYN deamination by clorgyline and harmine and (2) the reversal of the relative inhibitory effectiveness of the two classes of MAO inhibitors, (a) clorgyline and harmine and (b) pargyline, toward the three substrates. Semicarbazide did not inhibit the oxidation of any of the substrates. This indicates that the mitochondrial enzyme activity from the uterus, as in the liver, is a true monoamine oxidase.     

Observations on the inhibition of rat liver monoamine oxidase by clorgyline

Two forms of monoamine oxidase (MAO) denned as MAO A and B by others differ in their specificities to substrates and their sensitivities to the irreversible inhibitor clorgyline. From studies using the substrates 5-HT, tyramine and benzylamine, the presence of both MAO forms in rat liver mitochondria has been confirmed and some characteristics of their inhibition by varying concentrations of clorgyline investigated. Although both MAO forms showed time-dependent inhibition, this process occurred, in general, at a qualitatively slower rate for MAO B, despite the fact that this enzyme form requires higher concentrations of clorgyline than MAO A for inhibition of its activity. However, factors such as the concentration of enzyme, the concentration of clorgyline and the enzyme: drug ratio employed in the assay all influence the resultant time-course and the final degree of the inhibition observed. The possible importance of the lipid environment of the outer mitochondrial membrane in generating multiple MAO forms and in regulating the inhibition kinetics of these forms is discussed. The results indicate that the effects of pre-incubation time and the enzyme: drug ratio on inhibition of MAO by clorgyline should be fully recognized when using the drug to indicate multiple forms in animal tissues.     

Characterization and quantitation of monoamine oxidases A and B in mitochondria from human placenta

Monoamine oxidases (MAOs) A and B, flavin-containing enzymes found in the outer mitochondrial membrane, oxidize many important biogenic and xenobiotic amines. The two enzymes are expressed in many tissues, with some tissues containing primarily one form and others containing both. Although MAO in placental mitochondria is widely reported to be type A, some investigators have reported low levels of MAO B activity as well. Because placenta is considered the preferred source for purification of type A MAO, we have reinvestigated placental MAO by immunoblotting with monoclonal antibodies and active site labeling with the MAO-specific ligand [3H]pargyline. We have confirmed that placental mitochondrial preparations contain MAO A and low but significant MAO B catalytic activity, as judged by accepted pharmacological criteria (deprenyl-sensitive beta-phenylethylamine and benzylamine oxidation). Immunoblotting revealed polypeptides of sizes expected for both MAO A and B subunits in preparations of placental mitochondria, as well as in preparations of MAO A purified extensively from placenta by partitioning between dextran and polyethylene glycol polymers and chromatography on DEAE-Sepharose CL-6B. Both MAO A and B active sites could be quantitated in placenta by labeling mitochondrial preparations with the MAO-specific affinity ligand [3H] pargyline, followed by immunoprecipitation with MAO A- and MAO B-specific monoclonal antibodies. These results indicate that MAO B activity and protein is consistently present in mitochondrial preparations of human placenta.     

Phentermine inhibition of recombinant human liver monoamine oxidases A and B

Recent studies with rat tissue preparations have suggested that the anorectic drug phentermine inhibits serotonin degradation by inhibition of monoamine oxidase (MAO) A with a K(I) value of 85-88 microM, a potency suggested to be similar to that of other reversible MAO inhibitors (Ulus et al., Biochem Pharmacol 2000;59:1611-21). Since there are known differences between rats and humans in substrate and inhibitor specificities of MAOs, the interactions of phentermine with recombinant human purified preparations of MAO A and MAO B were determined. Human MAO A was competitively inhibited by phentermine with a K(I) value of 498+/-60 microM, a value approximately 6-fold weaker than that observed for the rat enzyme. Phentermine was also observed to be a competitive inhibitor of recombinant human liver MAO B with a K(I) value of 375+/-42 microM, a value similar to that observed with the rat enzyme (310-416 microM). In contrast to the behavior with rat tissue preparations, no slow time-dependent behavior was observed for phentermine inhibition of purified soluble human MAO preparations. Difference absorption spectral studies showed similar perturbations of the covalent FAD moieties of both human MAO A and MAO B, which suggests a similar mode of binding in both enzymes. These data suggest that phentermine inhibition of human MAO A (or of MAO B) is too weak to be of pharmacological relevance.     

In vivo and in vitro effects of amrinone and milrinone on hepatic xenobiotic metabolism in rats

Studies were performed on the response of hepatic xenobiotic metabolizing enzymes to in vitro and in vivo exposure to amrinone and milrinone, two new inotropic compounds used in congestive heart failure. Both drugs exerted selective effects on various cytochrome P-450-dependent metabolic activities as well as conjugating pathways. Aminopyrine N-demethylation was selectively inhibited by in vitro addition of milrinone but not amrinone, and laurate hydroxylation was inhibited by both drugs. Cytosolic glutathione-S-transferase activity was profoundly inhibited by in vitro addition of both drugs. In vivo administration of either drug did not lead to significant inhibition of the pathways studied other than laurate hydroxylation which was depressed 20-30%. Irreversible binding of [14C]-amrinone-derived radioactivity to microsomal protein was partially NADPH-dependent. Inhibition by SKF 525-A, alpha-naphthoflavone and various antioxidants was observed. No binding of [14C]-milrinone-derived radioactivity was seen. It is suggested that amrinone may selectively inhibit certain hepatic drug-metabolizing enzymes through metabolic electrophilic intermediates.     

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     

Evidence for existence of A and B form monoamine oxidase in mitochondria from dog platelets

It is known that platelet MAO appears to behave more like the B-form enzyme than the A-form enzyme based on inhibitor sensitivity and substrate specificity. However, dog platelets showed a different substrate specificity such as high activity with 5-HT and beta-PEA as substrates. Moreover, dog platelet MAO was sensitive to the drugs clorgyline and harmaline with 5-HT as the substrate, while it was sensitive to the drug deprenyl with beta-PEA as the substrate. These results also indicate the existence of two forms of MAO in dog platelets unlike in other platelets such as those from humans. A-form MAO from dog platelets was more stable against heat treatment at 55 degrees C than A-form MAO from dog liver and brain. On the other hand, there was no difference in the heat resistance of the three enzymes with beta-PEA as the substrate. After digestion with trypsin at 37 degrees C for 30 min, it was found that MAO from dog platelets, brain and liver were mostly inhibited with 5-HT as the substrate. In contrast, MAO in brain and liver were inhibited about 10%, but platelet MAO was inhibited about 50% with beta-PEA as the substrate. From these results, it is considered that dog platelet MAO exists as the two forms of the enzyme and has different enzymic properties in comparison with those of MAO from dog liver and brain mitochondria.     

The dose of nicorandil in men predicted from the inhibition of MAO activity by organic nitrates

The effect of N-(2-hydroxyethyl)-nicotinamide nitrate ester (nicorandil) on rat heart monoamine oxidase (MAO) was investigated in vitro. MAO activity was assayed by the isotopic method with 14C-tryptamine as substrate. Nicorandil significantly inhibited MAO activity of rat heart mitochondria. The effect of nicorandil on the enzymatic kinetics of MAO of rat heart mitochondria indicates that the anti-anginal drug acts as a competitive inhibitor of MAO. The inhibitor constant (Ki) of nicorandil was 4.93 mmol/l. The Ki of nicorandil was fitted to the formula derived from the relationship between the Ki values of glyceryl trinitrate, erythrytol tetranitrate, mannitol hexanitrate and isosorbide dinitrate and the dose of these drugs recommended to relieve anginal pain in patients. The dose of nicorandil was 0.071 mmol (15.0 mg at a time). This value agreed with the clinical dose for patients (10--20 mg at a time).     

Microsomal enzymes and potentiation of tyramine pressor response

Therapy with monoamine oxidase (MAO) inhibitors has produced hypertensive crises in patients who have ingested tyramine in food. This effect has been attributed to the inability of inhibited MAO to degrade tyramine, but recent work suggests that inhibition of hepatic microsomal oxidative enzymes may also be involved. In our experiments, SKF-525A was more potent (1000 times) than phenelzine as an inhibitor of microsomal tyramine hydroxylase, but less potent than phenelzine in potentiating the pressor response of tyramine. As SKF-525A was also shown to inhibit MAO (10 times less potent than phenelzine), it is suggested that inhibition of tyramine hydroxylase is not a major factor in potentiating the pressor response. In animals in which the microsomal enzymes were induced with phenobarbital, the pressor response to tyramine was not reduced, as would be expected if microsomal enzymes were regulating tyramine levels. These experiments suggest that tyramine potentiation is probably not a problem in therapy with drugs known to be microsomal enzyme inhibitors if these drugs have no MAO inhibitory activity.