Multiplicity of monoamine oxidase: inhibition of mitochondrial monoamine oxidase activity by isopropylhydrazide of D,L-serine]

Isopropylhydrazide of D,L-serine (IHS) inhibits by 50% (at 37 degrees for 10 min) deamination of serotonin or beta-phenylethylamine by monoamine oxidases from bovine brain stem mitochondrial membranes at the 2.6 X X 10(-5) M or 9 X 10(-5) M, respectively. In order to inhibit by 50% the deamination of tyramine under the same conditions a considerably lower (2.5 X X 10(-6) M) concentration of IHS is required. Kinetic studies of inhibition of enzymatic deamination of all the three biogenic monoamines by IHS showed that the irreversible blocking of the monoamine oxidase activity is preceeded by formation of dissociating enzyme-inhibitor complexes. Values of the dissociation constants of these complexes measured (at 37 degrees) with serotonin, phenylethylamine or tyramine as substrates for estimation of the residual monoamine oxidase activity are 0.47; 0.13 or 0.023 mM, respectively. Significant differences are also found between thermodynamic and activation parameters characterizing both both steps of interaction between IHS and the monoamine oxidases of mitochondrial membranes in the experiments with serotonin, phenylethylamine or tyramine as substrates. The data obtained suggest the existence of different monoamine oxidases (or their active sites) catalyzing oxidative deamination of serotonin, phenylethylamine or tyramine in the fragments of mitochondrial membranes from bovine brain stem.     

Effect of benzamide derivatives on rat brain monoamine oxidase activity in vitro

The ability of moclobamide and other benzamide derivatives to inhibit the activity of monoamine oxidase in the rat brain was studied. Distinct effects of these compounds on the deamination of serotonin and norepinephrine (MAO-A substrates); 2-phenylethylamine (selective MAO-B substrate); tyramine and dopamine (MAO-A and MAO-B substrates) are shown. It was demonstrated that among all the compounds studied moclobamide appeared to be the most active and selective inhibitor of MAO-A: at a concentration of 100 microM it caused a 100% inhibition of serotonin and norepinephrine deamination, which might be explained by the presence of C1 atom in the para-position of benzene ring in moclobamide molecule. Other benzamide derivatives were less active in inhibiting MAO-A and had but a negligible effect on dopamine- and 2-phenylethylamine deamination.     

Effects of antidepressive agents on glutamatergic autoregulatory presynaptic mechanism in the rat cerebral cortex

Amitriptyline, imipramine, desipramine, viloxazine, moclobemide and its derivative, novel antidepressant befol (10(-6)-5 x 10(-4) M) decreased by 12-20% K(+)-stimulated 3H-D-asp release from perfused synaptosomes of rat brain cortex. Glutamic acid diethyl ester (GDEE) (10(-4) M) antagonized the effect of amitriptyline, imipramine, desipramine and befol and reversed the effect of moclobemide and viloxazine. Among neuroleptics studied, only carbidine, which possesses antidepressant activity together with antipsychotic one in clinics, decreased 3H-D-asp release by GDEE-sensitive mechanism. Effect of haloperidol and chlorpromazine was not affected by GDEE. It is concluded that autoregulatory mechanism on the terminals of glutamatergic neurons may be involved in the antidepressant action.     

Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers

Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40-0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imipramine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.     

Inhibition of brain monoamine oxidase in the rat by multiple pyrazidol administration

Pyrazidol, which is chemically 2,3, 3a, 4, 5, 6-hexahydro-8-methyl-1H-pyrazino[3,2,1-j,k] carbazole hydrochloride (international name pirlindole) administered repeatedly (21 days) to rats at a dose of 25 mg/kg per os maintained the selectivity of its inhibitory effect toward type A MAO. When administered repeatedly the inhibitory effect of pyrazidol was 1.5-2-fold higher than after a single administration. The effect of pyrazidol on rat brain MAO was reversible whatever the route of administration. The enzymatic activity returned to normal within 24 h after the last administration. The data obtained suggest that the capacity of selective inhibiting the deamination of the neurotransmitters such as serotonin and noradrenaline in human brain is of paramount importance for therapeutic effect of pyrazidol.     

Different effects of monoamine oxidase inhibition on MPTP depletion of heart and brain catecholamines in mice

Pargyline, an inhibitor of monoamine oxidase type B (MAO-B), did not prevent the depletion of heart norepinephrine 24 hr after a single dose of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice. In mice killed 24 hr after the last of 4 daily doses of MPTP, the depletion of dopamine in the striatum and of norepinephrine in the frontal cortex was completely prevented by pargyline, but the depletion of heart norepinephrine was not prevented. These results with pargyline are the same as results obtained earlier with deprenyl, another selective inhibitor of MAO-B. The doses of pargyline and of deprenyl that were used resulted in almost complete inhibition of MAO-B activity (phenylethylamine as substrate) in brain, heart and liver of mice. Deprenyl did not inhibit MAO-A activity (serotonin as substrate) in brain, but pargyline caused some inhibition of MAO-A in brain. In heart and liver, serotonin was oxidized only at about 1/10 the rate of phenylethylamine oxidation, suggesting that MAO-B predominates in these tissues. Both pargyline and deprenyl caused some inhibition of serotonin deamination in heart and liver, suggesting that the oxidation may have been due partly to MAO-B. Experiments with selective MAO inhibitors in vitro showed that only about 20% of the oxidation of serotonin was occurring via MAO-B in heart and liver. The in vitro oxidation of MPTP by MAO in mouse brain, heart and liver was almost completely inhibited by pretreatment with either pargyline or deprenyl. Neither pargyline nor deprenyl had any significant effect on the concentrations of MPTP in brain or heart one-half hr after injection of MPTP into mice. The concentrations of the metabolite, MPP+ (1-methyl-4-phenyl-pyridinium), were markedly reduced in brain and in heart by pretreatment with either pargyline or deprenyl. The data suggest that MPP+ formation, which is necessary for the depletion of brain catecholamines after MPTP injection, may not be necessary for depletion of norepinephrine in heart. Since the oxidation of MPTP in vitro was inhibited more by pargyline or deprenyl pretreatment than was the appearance of MPP+ in vivo, the possibility exists that some MPP+ formation might occur by an enzyme other than MAO.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

Uptake of adrenaline by rat brain synaptosomes. Effects of several psychotropic drugs]

Comparative study of the uptake of 3H-epinephrine (3H-EN) and 3H-norepinephrine (3H-NE) into rat brain crude synaptosomes and effect of psychotropic drugs of different classes on this process showed that isolated nerve terminals had their own transport system for EN. The crude synaptosomal fraction had two transport system's for EN; high-specific active uptake with high affinity (KM = 3.7 + 0.21 microM) and low-affinity uptake (KM2 = 98.0 + 47.5 microM). En accumulation was saturable, stereo-specific and inhibited by ouabain (3 X 10(-3) M), protoveratrine A and B (10(-4) M), NaN3 (2 X 10(-3) M), 2,4-dinitrophenol (2 X 10(-3) M), p-chloromercuribenzoate (10(-4) M). Actinomycin D had no effect on the uptake of 3H-EN. 3H-HE was accumulated by two uptake system: 1-high affinity uptake system with KM values of 0.49 + 0.13 microM, 2-low affinity uptake system with KM values of 21.1 + 7.71 microM. Amphetamine, mesocarb, chlorpromazine, fluphenazine and haloperidol were equally effective inhibitors of 3H-EN and 2H-HE uptake. Imipramine, phenazepam, diazepam and carbamazepine (5 X 10(-5) M) had no effect on the uptake of 3H-NE. Imipramine, zimelidine, norzimelidine and viloxazine (5 X 10(-5) M) were more potent inhibitors of the 3H-EN uptake than that of 3H-NE.     

THE EFFECTS OF CHRONIC REGIMENS OF CLORGYLINE AND PARGYLINE ON MONOAMINE METABOLISM IN THE RAT BRAIN

The effects of chronic administration of clorgyline and pargyline on rat brain monoamine metabolism have been examined. The inhibitory selectivity of these drugs towards serotonin deamina-tion (MAO type A) and phenylethylamine deamination (MAO type B) can be maintained over a 21-day period by proper selection of low doses of these drugs (0.5-1.0 mg/kg/24h). The results are consistent with MAO type A catalyzing the deamination of serotonin and norepinephrine and with MAO type B having little effect on these monoamines. Dopamine appears to be dcaminated in vivo principally by MAO type A. Clorgyline administration during a 3-week period was accompanied by persistent elevations in brain norepinephrine concentrations; serotonin levels were also increased during the first 2 weeks, but returned towards control levels by the third week of treatment. Low doses of pargyline did not increase brain monoamine concentrations, but treatment with higher doses for 3 weeks led to elevations in brain norepinephrine and 5-hydroxytryptamine; at this time significant MAO-A inhibition had developed. The changes in monoamine metabolism seen at the end of the chronic clorgyline regimen are not due to alterations in tryptophan hydroxylase activity. At this time tyrosine hydroxylase activity was also unaffected.     

Brain region differences and some characteristics of monoamine oxidase type a and b activities in the vervet monkey

Monoamine oxidase in the vervet monkey showed greater variations in activity in six brain regions when tyramine or phenylethylamine was used as the substrate (3.8- to 4.1-fold differences) than when serotonin was the substrate (1.8-fold differences). With phenylethylamine and tyramine as substrates, the highest MAO specific activities were found in the hypothalamus and the lowest in the cerebellum and cortex. With serotonin as the substrate, the highest specific activities were in the mesencephalon and cortex. The inhibition of tyramine deamination by clorgyline and deprenyl yielded biphasic plots indicative of the presence of MAO-A and MAO-B enzyme forms in the vervet brain. On the basis of these inhibitor curves, the vervet brain could be estimated to contain approximately 85% MAO-B and 15% MAO-A, in contrast to rat brain which contains 45% MAO-B and 55% MAO-A. The inhibition of serotonin deamination by deprenyl in vervet brain yielded a biphasic plot, suggesting that some serotonin deamination in the vervet is accomplished by the MAO-B enzyme form. Estimations of the relative amounts of MAO-A and MAO-B based on inhibitor curves or based on substrate ratios yielded proportionate results which were in close agreement across the different brain regions, supporting the validity of these approaches to estimating MAO-A and MAO-B activities.     

Mechanism of the inhibiting effect of atypical antidepressants and psychostimulants on the synaptosomal uptake of monoamines

The effects of viloxazine and zimelidine on reverse monoamine neurotransmitter uptake by crude synaptosomal fraction of the rat brain were studied and compared to those of imipramine, amphetamine and cocaine. Imipramine noncompetitively inhibited the uptake of all the monoamines under study, with a greater specificity as regards serotonin. Viloxazine and zimelidine strongly inhibited the transport of noradrenaline, dopamine and serotonine, the transport of the latter being inhibited to a greater degree. Kinetic analysis showed the active centers of noradrenaline and dopamine carriers to be very much alike, those of catecholamine and serotonin carriers to be less alike. The data obtained made it possible to describe (at least partly) the structure of the active centers of monoamine carriers and to specify the action modes of antidepressants and psychostimulants.     

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.     

Inhibition of serotonin uptake in synaptosomes and glia cells by some pharmacological substances

The uptake of serotonin -14 C by glial cells and synaptosomes of the rabbit brain cortex was studied. The Km value of the uptake of serotonin -14 C proved to be equal (0.83 + 0.02 microM) both for synaptosomes and glial cells. Synaptosomes of the rabbit brain cortex take up serotonin -14 C twice as fast as glial cells (uptake rates were compared from protein). Among psychotropic drugs studied the tricyclic antidepressant imipramine and psychostimulant cocaine turned out the most active inhibitors of both synaptosomal and glial uptake of serotonin -14 C. The drugs in 50 microM concentration inhibit the uptake of serotonin -14 C in synaptosomes and glial cells by 90 and 75-80%, respectively.     

Phenothiazines and structurally related compounds as inhibitors of adenosylmethionine decarboxylase: effects on the purified enzyme

The effects of chlorpromazine, imipramine, thioridazine, chlorprothixene, amitriptyline, desipramine and triflupromazine on adenosylmethionine decarboxylase purified from rat liver have been studied. The compounds caused competitive inhibition of the enzyme at 10(-5) - 10(-3) M concentrations. For chlorprothixene and triflupromazine the inhibition was linear, while the other drugs showed increasing, nonlinear inhibition at higher concentrations. Apparent Ki's for the compounds were between 6.8 X 10(-5) M (for chlorprothixene) and 6.4 X 10(-4) M (for desipramine). Inhibition of 50% under optimal assay conditions was achieved between drug concentrations of 1.3 X 10(-4) M (thioridazine) and 1.3 X 10(-3) M (imipramine).     

Effect of dihydroxyphenylalanine, imipramine and coffeine on the light induced decrease in nocturnal serotonin N-acetyltransferase in the rat pineal gland.

The s.c. administration of 150mg L-dihydroxphyenylalaine/kg b.w. 15 min before the decapitation prevents the light induced decrease in nocturnal serotonin N-acetyltransferase activity in the rat pineal gland. The s.c. administration of 50mg imipramine/kg b.w., resp. 100mg/kg b.w., 15 min before the decapitation, slows down, or prevents the light induced fall in the activity. The maintenance of a sufficient level of active norepinephrine on beta-receptors, either by displacement of norepinephrine in the nerve endings by dopamine, or by the inhibition of norepinephrine reuptake by imipramine, thus slows down or prevents the decrease in serotonin N-acetyltransferase activity after exposure to light during the night. The i.p. administration of a phosphodiesterase inhibitor coffeine citrate in a dose 200mg/kg 90 min after switching off the light for the night stimulated serotonin N-acetyltransferase activity 270 min after the light and been switched off, but did not influence the abrupt decrease induced in nocturnal activity by exposure to light.     

Effects of tandamine and pirandamine,selective blockers of biogenic amine uptake mechanisms,on gastric acid secretion and ulcer formation in the rat

Tandamine and pirandamine and various structurally-related compounds, which were known to inhibit the norepinephrine and/or serotonin uptake mechanisms, lack central anticholinergic activity and differ chemically from the known tricyclic antidepressant drugs, were examined for their effects on gastric acid secretion and ulcer formation in the rat. Tandamine and its structurally-related compounds, but not pirandamine or its structurally-related compound, given intraperitoneally, inhibited gastric acid secretion and were similar in activity to imipramine. Like imipramine, tandamine was effective when given perorally. None of the compounds examined, administered intraperitoneally, were effective in reducing the ulcer formation in 19 h pylorus-ligated animals, while imipramine was effective. Tandamine, like imipramine, inhibited ulcer formation in 10 h pylorus-ligated animals and in 19 h pylorus-ligated animals when given in divided doses. Tandamine and its structurally-related compounds, but not pirandamine or its structurally-related compound, given subcutaneously, prevented reserpine-induced gastric ulceration; imipramine was also effective. Tandamine and imipramine, administered intraperitoneally, prevented cold-restraint gastric ulceration. The compounds which block the norepinephrine, or in addition the serotonin, uptake mecahnism, but not those which block only the serotonin uptake mechanism, inhibited the gastric acid secretion and reserpine-induced ulceration. Thus, these latter activities appear to be correlated with the inhibition of the norepinephrine, rather than serotonin uptake mechanism.     

Deamination of indolalkylamines in hyperthermia

The experiments on dogs subjected to the outward overheating allow concluding that the quantities of deaminated serotonin in mitochondrial fractions of the brain stem, heart muscles and liver increase twice as compared with the normal levels. Particularly, in the liver the value of Km of this process is equal to 1.6 x 10(-3) M, while without the overheating it is equal to 3.6 x 10(-3) M. Overheating makes the process of tryptamine deamination in mitochondrial fractions of the heart and liver more intensive. However, overheating causes a higher increase of the monoaminooxidase activity with respect to serotonin than with respect to other substrates.     

Inhibition of Monoamine Oxidase by 7-Chloro-4-Nitrobenzofurazan

7-Chloro-4-nitrobenzofurazan (NBD-Cl) is a potent inhibitor of both types of monoamine oxidase (MAO). NBD-Cl competitively inhibited the oxidative deamination of kynuramine catalyzed by human placenta MAO-A, the oxidative deamination of benzylamine catalyzed by bovine liver MAO-B, the oxidative deamination of serotonin catalyzed by rat brain MAO-A, and the oxidative deamination of phenylethylamine catalyzed by rat brain MAO-B. In addition, a time-dependent inactivation of MAOs by NBD-Cl has been demonstrated upon incubation of the enzyme preparations with NBD-Cl at pH 9, but not at pH 7.5. The time-dependent inhibition of MAO by NBD-Cl could be prevented by the addition of 4-nitrophenyl azide, an active site-directed label of MAO, during incubation of the enzyme with NBD-Cl. On the basis of these findings, it is suggested that at pH 9, NBD-Cl modifies one (or more) essential lysine residue(s) in the active sites of the two types of MAO.     

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%.     

Effect of prolonged cold exposure on monoamine oxidase activity and kinetics and on serotonin metabolism in the rat brain

Effects of long-term cold exposure on the content of serotonin and its metabolite 5-hydroxyindolacetic acid (5-HIAA) and monoamine oxidase (MAO) activity and kinetic parameters (Km and Vmax) of oxidative deamination of serotonin in rat brain stem. The increase of 5-HIAA level in the initial period of chronic cold exposure was determined by the blockade of active metabolite transport from the brain. The level of serotonin and the rate of its catalytic deamination by MAO were found to be decreased in cold-adapted rats. The magnitude of the Km of serotonin deamination was unchanged.