Effect of 6-aminonicotinamide on monoamine oxidase and Na+K+ATPase activity in different regions of rat brain

The monoamine oxidase activity in the cerebral hemispheres decreased significantly after 2, 4, 8 and 16 hr of 6-amino-nicotinamide (35 mg/kg body weight, i.p.) administration. In the cerebellum, the MAO activity was not affected significantly. In the brain stem, however, a significant increase was observed after 2 hr of drug administration followed by a gradual decrease at later time intervals. The Na+K+ATPase activity in the cerebral hemispheres was increased significantly at 2 and 4 hr of 6-aminonicotinamide administration. This was followed by a gradual decrease at later time intervals. In the cerebellum, like monoamine oxidase, the Na+K+ATPase did not change significantly. The brain stem showed a decrease at 2 hr of drug administration, followed by a significant increase at 4 hr and then a gradual decrease to near control values.     

Binding and deamination of various substrates by types A and B monoamine oxidase in bovine brain mitochondria

The binding and deamination of four substrates by type A and type B monoamine oxidase (MAO) in bovine brain mitochondria were investigated in mixed substrate experiments. MAO activity in bovine brain mitochondria, with 5-hydroxytryptamine (5-HT) as substrate, was highly sensitive to clorgyline and less sensitive to deprenyl, while MAO activity with benzylamine or β-phenylethylamine (PEA) as substrate was highly sensitive to deprenyl and less sensitive to clorgyline. On the other hand, when tyramine plus PEA was used as substrate, the inhibition curves of clorgyline and deprenyl were both biphasic. These results indicate that 5-HT and benzylamine were preferentially deaminated by type A MAO and type B MAO, respectively, and that tyramine and PEA were deaminated by both types of MAO. Studies on the inhibition by clorgyline plus deprenyl of tyramine deamination (in the absence and presence of another substrate) showed that the deamination of tyramine by both type A and type B MAO was inhibited by PEA or benzylamine, while only type A MAO was inhibited significantly by 5-HT. The KA_i value, the dissociation constant of the type A MAO and 5-HT complex, and the KB_i values, the dissociation constants of the type B MAO and PEA or benzylamine complex, were almost equal to the K_m values of type A MAO and type B MAO respectively. The KA_i values for PEA and benzylamine were 78 and 58 μM respectively. For the type B MAO-5-HT complex, the dissociation constant KB_i was 1447 μM. These results show that type A MAO deaminates tyramine and 5-HT whereas benzylamine is not deaminated, but only binds to the substrate binding site of type A MAO with almost the same rate as that for deamination by type B MAO; with type B MAO, tyramine, PEA and benzylamine are deaminated, whereas 5-HT is not deaminated and binds to the substrate binding site of type B MAO with low affinity.     

Characterization of N-methylphenylethylamine and N-methylphenylethanolamine as substrates for type A and type B monoamine oxidase

N-Methylphenylethylamine (MPEA) and N-methylphenylethanolamine (MPEOA) were characterized as substrates for type A and type B monoamine oxidase (MAO) in rat brain mitochondria. The inhibition experiments with clorgyline and deprenyl showed that the inhibition patterns with MPEA as substrate were dependent on substrate concentrations but that this amine was a common substrate for both types of MAO at all substrate concentrations tested. When MPEOA was used as substrate, the inhibition patterns differed markedly at different substrate concentrations; at 10.0 /smM, MPEOA acted as a specific substrate for type B MAO, but at 100 and 1000, μM it became a common substrate for both types. Kinetic analyses were carried out for MPEA and MPEOA with the uninhibited, the clorgyline-treated (type B MAO), and the deprenyl-treated enzyme (type A MAO). With the uninhibited enzyme, there were downward deflections in the curves of Lineweaver-Burk plots for both MPEA and MPEOA, suggesting the existence of different affinity components derived from type A and type B MAO. By means of the double-reciprocal plots, using the clorgyline- and the deprenyl-treated enzyme, it was confirmed that the high affinity corresponded to that for type B MAO and the low affinity to that for type A MAO for both MPEA and MPEOA. Therefore, the changes in the inhibition pattern at different substrate concentrations may be due to different affinities of the substrate for both types. By comparing the K_m and V_max values of both types observed for MPEA and MPEOA, it was pointed out that the β-hydroxylation of MPEA tended to increase the K_m value for type A MAO and to decrease the V_max values for both types.     

Characterization of eight biogenic indoleamines as substrates for type A and type B monoamine oxidase

Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, 5-hydroxytryptamine (5-HT), 5-hydroxy-N-methyltryptamine, bufotenine, 5-methoxytryptamine, and 5-methoxy-N,N-dimethyltryptamine were characterized as substrates for type A and type B monoamine oxidase (MAO) in rat liver mitochondria. Experiments on sensitivity to clorgyline and to deprenyl, using two substrate concentrations, showed that tryptamine and its N-methylated and N,N-dimethylated derivatives were common substrates for both types of MAO at a substrate concentration of 20.0 μM; at 1000 μM, tryptamine and N-methyltryptamine were common, but N,N-dimethyltryptamine became specific for type B MAO. All the 5-hydroxy- or 5-methoxy-indole derivatives were almost completely specific for type A MAO at a substrate concentration of 20.0 μM; when the concentration was 1000 μM, some of the MAO activity was due to type B MAO for 5-HT, bufotenine and 5-methoxytryptamine. The rat liver mitochondrial enzyme was pretreated with 10^?7M clorgyline and 10^?7M deprenyl to obtain, respectively, the type B-rich and the type A-rich enzyme. These enzyme preparations were subjected to kinetic analyses for the eight amines. From the kinetic analyses, together with data on inhibitor sensitivity, the following phenomena can be described. N-Methylation of tryptamine or of 5-HT did not cause appreciable changes in the specificity of the substrates toward each type of MAO, but it elevated the K_mvalue of type B MAO when the values for tryptamine and N-methyltryptamine were compared. N,N-Dimethylation of tryptamine and 5-HT tended to increase the specificity for type B MAO. All the dimethylated compounds had very low activities with either type A or type B MAO. Either the 5-hydroxy- or the 5-methoxy-group contributed to the specificity of the substrates for type A MAO.     

Phenylhydrazine: Selective inhibition of human brain type B monoamine oxidase

Phenylhydrazine (PhNNH₂) was found to irreversibly inhibit both human brain type A and type B monoamine oxidase (MAO) in vitro. PhNNH₂ was a more effective inhibitor of the B form of the oxidase in that 50 per cent inhibition of phenylethylamine (PEA) deamination occurred at approximately 4 μM, whereas the equivalent inhibition of 5-hydroxytryptamine (5-HT) deamination required almost 200 μM of the inhibitor. Similarly, inhibition of PEA deamination occurred at a faster rate than that of 5-HT metabolism. Evidence is presented that suggests that PhNNH₂ inhibits both forms of MAO by two distinct mechanisms, a temperature-sensitive and a temperature-insensitive process. The temperature-insensitive component proceeded extremely rapidly, compared to the temperature-dependent component. For example, at 4° inhibition of PEA deamination by PhNNH₂ attained its maximum within a 30-sec preincubation period. Both inhibition processes were diminished when PhNNH₂ was preincubated in a nitrogen atmosphere compared to that in air. Fifty per cent inhibition of PEA and 5-HT deamination by the PhNNH₂ temperature-insensitive component occurred at 20 μM and 1 mM respectively. The presence of two distinct inhibition processes is consistent with prior studies that have demonstrated that more than 1 mole of PhNNH₂ is bound per mole of MAO.     

Regional inhibitory effect of ethanol on monoamine synthesis regulation within the brain

Catecholamine biosynthesis from labeled tyrosine was examined in hippocampal and olfactory tubercle slices. The presence of depolarizing concentrations of potassium (25–55 mM) resulted in significant increases in catecholamine synthesis in both tissue preparations. Following depolarization of slices in 25 mM potassium, the major portion of newly synthesized catecholamine was retained in the tissue whereas incubation in 55 mM potassium resulted in a significant portion of the newly synthetized catechols being released into the media. Addition of ethanol in concentrations of 0.2–0.8% (w/v) to tissue slices prepared from brain regions rich in dopaminergic nerve terminals, such as olfactory tubercles, caused a dramatic reduction in dopamine synthesis from labeled tyrosine induced by potassium depolarization. The ability of ethanol to inhibit the potassium-induced acceleration of catecholamine biosynthesis appeared to be related to the dopaminergic innervation of the tissue. Catecholamine synthesis in slices of depolarized hippocampi, tissue which is relatively free from substantial dopaminergic innervation, was unaffected by ethanol in concentrations as high as 1.2% (w/v). These results are discussed in terms of the selectivity and mechanisms of the ethanol-induced effects.     

Monoamine oxidase in human platelets: Kinetics and methodological aspects

The kinetic properties of human platelet monoamine oxidase (MAO) were examined in 20 apparently healthy controls. The mean value (±S.D.) of the maximum velocity (V) was found to be 5.36 ± 1.97 pmoles of product formed/10° platelets/min and the Michaelis-Menten constants were for phenethylamine (K^PEA_m) 14.6 ± 8.20 μM and for oxygen (K_m^O₂)254 ± 125 μM, when assayed in 0.1 M phosphate buffer, pH 7.4. The relation between the value of the corresponding apparent constants was studied. Inhibition of the enzyme activity was seen at 20 μM of PEA and 180 μM of oxygen. The enzyme kinetics were also studied at different pH. Two pK values were found, pK₁ = 6.65 and pK₂ = 6.95. The influence of homogenization on the MAO activity was compared with the activity in the undisrupted platelet. At PEA concentrations below 10 μM higher MAO activities were found in the intact cell. A 15 per cent loss of activity was detected in platelet samples after storing at ?20° for three and a half years.     

N-[2(o-iodophenoxy)ethyl]cyclopropylamine hydrochloride (LY121768), a potent and selective irreversible inhibitor of type a monoamine oxidase

The effects of N-[2-(o-iodophenoxy)ethyl]cyclopropylamine hydrochloride (LY121768) on types A and B monoamine oxidase (MAO) assayed with radiocarbon-labeled serotonin and phenyl-ethylamine, respectively, were studied in vitro and in vivo. Type A MAO from rat brain was inhibited in vitro by LY121768 with an ic₅₀ of 4 × 10^?10 M, whereas 2500 times higher concentrations of LY121768 $\text{(}\text{ic}{}_{50}\text{= 1 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ were required to inhibit type B MAO. The inhibition of type A MAO increased with time of incubation of LY121768 with enzyme prior to substrate addition and persisted after dialysis, indicative of irreversible inhibition. The irreversible inactivation was prevented by harmaline, a reversible, competitive inhibitor of type A MAO, indicating a requirement for catalytic activity of MAO in the time-dependent inactivation by LY121768. In rats, LY121768 selectively inhibited type A MAO in brain and in liver at low doses. The inhibition of type A MAO persisted for several days after a single 10 mg/kg i.p. dose of LY121768 and was associated with a significant increase in brain dopamine, norepinephrine and epinephrine concentrations and a significant decrease in the concentration of the dopamine metabolites, homovanillic acid and 3.4-dihydroxyphenylacetic acid. The inactivation of type A MAO by LY121768 in vivo was prevented by co-administration of harmaline, indicating a similar mechanism for the in vivo inactivation as for the in vitro inactivation of MAO by LY121768. A reasonable inference from these data and from previous literature is that LY121768 acts as a “suicide substrate” for MAO and inactivates the enzyme by formation of a reactive intermediate which binds covalently to the enzyme. The presence of iodine in the LY121768 molecule not only confers high selectivity for type A MAO but offers a site for radionuclide introduction that might be a useful means of labeling type A MAO in vitro or in vivo for various purposes.     

Inhibition of rabbit liver monoamine oxidase by nitro aromatic compounds

Nitrobenzoid, nitroheterocyclic and cyanobenzoid compounds inhibit type B monoamine oxidase. A partially purified enzyme preparation from rabbit liver mitochondria, oxidizing rho-dimethyl-aminobenzylamine as the substrate, was competitively inhibited by nitrobenzoid compounds with K1 values in the range of 0.28 muM for rho-dinitrobenzene to 0.56 muM for rho-nitrobenzoic acid. The potencies of nitrobenzoid compounds were positively correlated with the Hammett sigma value for each substituent on nitrobenzene. Dinitro derivatives were slightly more potent than the corresponding mononitro compounds but not as potent as would be expected from their sigma values. For the nitroheterocyclic compounds, inhibition was also competitive; the lowest K1 was 1.3 muM for 5-nitrofurfural semicarbazone (nitrofurazone). Compounds with cyano groups in place of nitro groups were also inhibitory; the most potent was rho-acetobenzonitrile with a K1 of 1.3 muM. The results of this study indicate that, in addition to nitrobenzoid compounds, other compounds with planar, electron-deficient nuclei are effective inhibitors of type B monoamine oxidase. Although hydrophobic and steric parameters may play some role in inhibition, the predominant factor is the electron-withdrawing power of the ring substituents.     

Time-dependent inhibition of monoamine oxidase by β-phenethylamine

Several reports have suggested that monoamine oxidase activity towards β-phenethylamine is inhibited by high concentrations of that substrate. This inhibition is not found if initial velocities are measured, but there is a slower time-dependent inhibition at higher β-phenethylamine concentrations. Such time-dependent inhibition is not found with tyramine as substrate or upon incubation of the enzyme with the reversible inhibitor amphetamine. The inhibition is not due to the accumulation of phenacetaldehyde, phenylethanol or phenacetic acid, or to a reaction of any of these three products either with each other or with β-phenethylamine. Although the inhibition is time-dependent, the inactivated enzyme slowly regains activity upon removal of the β-phenethylamine. A model is proposed to explain the observed inhibition.     

Some properties of monoamine oxidase and a semicarbazide sensitive amine oxidase capable of the deamination of 5-hydroxytryptamine from porcine dental pulp

The deamination of 5-hydroxytryptamine, tryptamine and benzylamine by porcine dental pulp membrane preparations is brought about not only by monoamine oxidase, but also by a clorgyline (and deprenyl) resistant, semicarbazide sensitive enzyme. The semicarbazide sensitive enzyme was also inhibited by aminoguanidine, hydroxylamine and phenylhydrazine, but was not affected to any significant extent by incubation at 50° for up to 100 min. There was, on the other hand, considerable inhibition of monoamine oxidase activity after incubation at this temperature. The semicarbazide sensitive enzyme neither metabolised, nor was inhibited by putrescine or cadaverine. Mixed substrate experiments indicated that 5-hydroxytryptamine and tryptamine interacted at the same catalytic centre on the semicarbazide sensitive enzyme.