Release of 3H-5-hydroxytryptamine by amiflamine and related phenylalkylamines from rat occipital cortex slices.

In the present study amiflamine and other related reversible monoamine oxidase-A (MAO-A) inhibitory phenylalkylamines were examined in vitro for their ability to induce release of 3H-5-hydroxytryptamine (3H-5-HT) from rat occipital cortex slices. The slices were preincubated with 3H-5-HT 0.1 mumol/l in the presence of the irreversible MAO inhibitor pargyline 50 mumol/l and then continuously superfused. The effects were compared with those of the 5-HT releaser p-chloroamphetamine (pCA), the reversible MAO-inhibitor alpha-ethyltryptamine and the 5-HT uptake inhibitor citalopram. Amiflamine, some related compounds and alpha-ethyltryptamine which in vivo after transport by the 5-HT uptake mechanism preferentially inhibit MAO within the serotonergic neurons caused a Ca2+-independent release of 3H-5-HT. Some transported compounds, particularly NBF 027 were, however, very weak releasers of 5-HT. This release and that induced by pCA was prevented by citalopram in the superfusion medium. FLA 365, FLA 417 and FLA 1088, which are not transported into the neurons, were poor releasers of 5-HT. It is concluded that compounds which were effective releasers of 5-HT in vitro were those that are transported into the serotonergic neurons by the 5-HT carrier in vivo and has in addition an ability to mobilise vesicular 5-HT.

Inhibition of monoamine oxidase within monoaminergic neurons in the rat brain by (E)-beta-fluoromethylene-m-tyrosine (MDL 72394).

The irreversible inhibition of the monoamine oxidase (MAO) activity within monoaminergic neurons in the rat brain 24 h after single or repeated administration of (E)-beta-fluoromethylene-m-tyrosine (FMMT, MDL 72394) was examined. The enzyme activity was determined by incubating synaptosome-rich homogenates of hypothalamus or striatum with low concentrations of 5-[14C]hydroxytryptamine (5-HT), [14C]noradrenaline (NA), or [14C]dopamine (DA) in the absence and presence of the selective amine uptake inhibitors citalopram (5-HT), maprotiline (NA), and GBR 12909 (DA). After a single subcutaneous injection of FMMT, the inhibition of MAO within the noradrenergic and dopaminergic neurons was significant but only slightly greater than that outside these neurons. The opposite relationship was observed for the serotonergic neurons. After 7 days' treatment of rats with carbidopa, 20 mg/kg p.o., + FMMT once daily, the preference for the inhibition of MAO within the noradrenergic and dopaminergic neurons was accentuated further. The inhibition outside the serotonergic neurons was still greater than within these neurons. The NA uptake inhibitor CPP 199 antagonized the selective inhibition of MAO within the noradrenergic neurons, which indicates that this preference is due to the accumulation of the active metabolite (E)-beta-fluoromethylene-m-tyramine by the NA transporter.

Postmortem- and cryostability of the potassium-evoked release of [3H]5-hydroxytryptamine from rat cerebral cortical miniprisms.

A prerequisite for the study of neurotransmitter release from human brain autopsy samples with histories of different diseases is that the cryo- and postmortem stability of the release process is good. In the present study, the effect of post-mortem delay and of storage at -70 degrees C by the "slow freeze--fast thaw" method of Hardy et al. [J Neurochem (1983) 40: 608-614] (which allows for the retention of metabolic activity of the tissue after the storage and thawing) of rat cerebral cortex samples upon the release of [3H]5-hydroxytryptamine ([3H]5-HT) from prelabelled miniprisms has been investigated. Storage of samples at -70 degrees C by this method resulted in samples that accumulated less [3H]5-HT but showed an increased sensitivity to the Ca2+-dependent releasing properties of K+ when compared with "fresh" samples. On the other hand, the sensitivity of the K+-evoked release to the inhibitory effects of the serotoninergic agonist 5-methoxy-N,N-dimethyltryptamine were reduced by storage. The effects on [3H]5-HT accumulation and on K+-evoked release were due mainly to the freeze-thaw procedure, the length of storage at -70 degrees C having only a minor influence on these parameters. A post-mortem interval of 5 hours at either +4 or +22 degrees C prior to storage of the tissue reduced the K+-evoked release of tritium, but did not affect the accumulation of [3H]5-HT or the inhibitory effects of 5-methoxy-N,N-dimethyltryptamine on the K+-evoked release over and above the effects produced by the storage per se.

Selective inhibition by 4,alpha-dimethyl-m-tyramine (H77/77) and 4-methyl-alpha-ethyl-m-tyramine (H75/12) of the monoamine oxidase within serotonergic and noradrenergic neurons in the rat brain.

1. The inhibition of monoamine oxidase (MAO) by 4, alpha-dimethyl-m-tyramine (H77/77) and 4-methyl-alpha-ethyl-m-tyramine (H75/12), two amine releasing compounds, within monoaminergic neurons in the rat hypothalamus and striatum in vivo was determined. This was performed by measuring the protection of MAO by the test compound against the irreversible inhibition produced by phenelzine. The MAO activity inside and outside monoaminergic synaptosomes in homogenates of brain tissue was measured in the absence and presence of selective uptake inhibitors at low concentrations of 14C-labelled 5-hydroxytryptamine, noradrenaline or dopamine. 2. It was found that H77/77 and H75/12 produced a pronounced protection against phenelzine within the serotonergic and noradrenergic neurons, whereas much less effect was observed outside these neurons. 3. It was shown that the protection by H75/12 within the serotonergic neurons was somewhat reduced in 5-HT depleted reserpinized rats and that the protection outside these neurons was abolished. Some of the protection of MAO might therefore have been brought about by 5-HT molecules released by H75/12. 4. The marked inhibition of MAO within serotonergic and noradrenergic neurons was counteracted by amine uptake inhibitors and is accordingly brought about by the high concentrations of the accumulated compounds. 5. In contrast to other neuron selective MAO inhibitors, H75/12 decreased the 5-HT concentration in the hypothalamus showing that the releasing effect dominated over the MAO inhibitory effect.

Selective monoamine oxidase inhibitors. 4. 4-Aminophenethylamine derivatives with neuron-selective action.

Nine 4-aminophenethylamine derivatives were synthesized and tested for monoamine oxidase (MAO) inhibitory effects with particular attention to their selectivity for MAO within monoaminergic neurons in the rat brain. All compounds selectively inhibited the A form of MAO in vitro. Some of the compounds inhibited the MAO within the monoaminergic neurons at much lower doses than those required for inhibition of MAO within other cells in vivo. The most potent compounds in this respect were 4-amino-2-fluoro-alpha-methylphenethylamine (5) and 4-amino-2-chloro-alpha-methylphenethylamine (4).

Failure to detect any transport of the irreversible monoamine oxidase inhibitor N-cyclopropyl-4-chloroamphetamine by the carrier of 5-hydroxytryptamine in the brain of the rat in vivo.

N-Cyclopropyl-4-chloroamphetamine (LY 93716) was examined for its potential preference for inhibiting the activity of monoamine oxidase (MAO) within serotonergic nerve terminals in the hypothalamus of the rat. Such an effect should support the hypothesis of Fuller and Perry (1977) (Neuropharmacology 16: 495-497) that this compound is transported by the uptake mechanism for 5-hydroxytryptamine (5-HT). By using a small (0.1 microM) substrate concentration of [14C]5-HT and a synaptosomal preparation incubated in the absence and presence of a selective inhibitor of the uptake of 5-HT (citalopram) it is possible to measure the activity of MAO in serotonergic nerve terminals. It was found that LY 93716 caused greater inhibition outside than inside the serotonergic nerve terminals when the activity of MAO was analysed 24 hr after the injection, but an inverted relationship was observed when analysed 72 hr after administration. Inhibition of uptake did not cause any change in the inhibition of MAO within the serotonergic nerve terminals at the former time but antagonized the inhibition observed 72 hr after the injection. It is concluded that the latter effect was due to antagonism of the neurotoxic action of LY 93716 and that no evidence was found that LY 93716 is transported by the uptake carrier for 5-HT.

Selective monoamine oxidase inhibitors. 3. Cyclic compounds related to 4-aminophenethylamine. Preparation and neuron-selective action of some 5-(2-aminoethyl)-2,3-dihydroindoles.

Nine 5-(2-aminoethyl)-2,3-dihydroindole derivatives were synthesized and tested as monoamine oxidase (MAO) inhibitors in vitro and in vivo. All compounds were found to be selective MAO-A inhibitors in vitro, the most active ones, 5-[1-(2-aminopropyl)]-2,3-dihydro-4-methylindole acetate (3), 5-[1-(2-aminopropyl)]-4-chloro-2,3-dihydroindole acetate (5), 5-[1-(2-aminopropyl)]-2,3-dihydro-1-ethyl-4-methylindole tartrate (6), 5-[1-(2-aminopropyl)]-2,3-dihydro-1-ethyl-6-methylindole tartrate (7), and 5-[1-(2-aminobutyl)]-4-chloro-2,3-dihydroindole acetate (9) being equipotent with amiflamine, (S)-(+)-4-(dimethylamino)-2, alpha-dimethylphenethylamine. Some of the compounds, 3, 6, 5-[1-(2-aminopropyl)]-2,3-dihydroindole acetate (1), and 5-[1-(2-amino-2-methylpropyl)]-2,3-dihydroindole acetate (8), were found to be very potent inhibitors of MAO in serotonergic and/or noradrenergic nerve terminals in the rat brain in vivo, inhibiting MAO within these neurons at doses 1/10 of those required to inhibit MAO in other neurons or cells. Compound 1 was also a potent and selective inhibitor of MAO within dopaminergic nerve terminals in vivo. This neuron selectivity is due to the uptake of these compounds by the neuronal uptake mechanisms.

A and B forms of monoamine oxidase within the monoaminergic neurons of the rat brain.

The inhibition of the A and B forms of monoamine oxidase (MAO) inside and outside serotonergic, noradrenergic, and dopaminergic synaptosomes in homogenates of rat hypothalamus or striatum by clorgyline, a selective and irreversible MAO-A inhibitor, and selegiline, a selective and irreversible MAO-B inhibitor, was examined. Intrasynaptosomal deamination at low concentrations of the substrates [14C]5-hydroxytryptamine ([14C]5-HT; 0.1 microM), [14C]noradrenaline (0.25 microM), [14C]3,4-dihydroxyphenylethylamine ([14C]dopamine; 0.25 microM), and [14C]tyramine (0.25 microM) was hindered by selective uptake inhibitors (citalopram, maprotiline, and amfonelic acid) in the incubation media. Thus, the difference between the deamination of 14C-amine in the absence and presence of the appropriate selective uptake inhibitor provided a measure of deamination in the specific aminergic synaptosomes. This was verified by determining the loss of MAO activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine and p-chloroamphetamine. Results with the two inhibitors revealed that the A and B forms were responsible for 80 and 20%, respectively, of the deamination of [14C]5-HT within serotonergic synaptosomes from the hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes from the hypothalamus and that of [14C]dopamine and [14C]tyramine within the striatal dopaminergic synaptosomes were due to MAO-A. About 10% of the deamination of [14C]noradrenaline, [14C]dopamine, and [14C]tyramine outside the noradrenergic or dopaminergic synaptosomes was brought about by the B form, with the remainder being deaminated by MAO-A.

Alpha 1-adrenoceptor function in the rat hippocampus as assessed by noradrenaline-stimulated inositol phospholipid breakdown after destruction of noradrenergic neurons by neonatal 6-hydroxydopamine treatment.

Neonatal 6-hydroxydopamine treatment of male Sprague-Dawley rats resulted in a large depletion of noradrenaline 84-87 days later, as demonstrated by an 82% reduction in the intra-synaptosomal deamination of [14C]noradrenaline within noradrenergic synaptosomes with only marginal effects upon the extrasynaptosomal deamination. The hippocampal stimulation of inositol phospholipid breakdown by noradrenaline was, however, unaffected by the neonatal 6-hydroxydopamine treatment.

Inhibition of monoamine oxidase in monoaminergic neurones in the rat brain by irreversible inhibitors.

The irreversible inhibition of monoamine oxidase (MAO) inside and outside monoaminergic neurones in the rat brain by the suicide inhibitors clorgyline, selegiline (l-deprenyl), pheniprazine, phenelzine, iproniazid, pargyline and the d- and l-enantiomers of tranylcypromine was determined. This was achieved by incubating crude synaptosomal preparations of hypothalamus and striatum from rats treated with the inhibitors 24 hr earlier, with low concentrations of [14C]serotonin (0.1 microM), [14C]-noradrenaline (0.25 microM) and [14C]dopamine (0.25 microM) in the absence and presence of selective uptake inhibitors (citalopram, maprotiline and amfonelic acid, respectively). It was found that all inhibitors inhibited the deamination of serotonin and noradrenaline outside the amine neurons at slightly lower doses than that within these neurones. This could at least in part be due to protection of MAO by the endogenous amines in these neurones. The deamination of dopamine was rather more strongly inhibited inside the neutrons than outside, particularly at higher doses. There was no indication that tranylcypromine or phenelzine was accumulated in the neurones by the membranous amine uptake mechanisms. The rate of the recovery of the deaminating activities inside and outside the serotonergic and noradrenergic neurones in hypothalamus after phenelzine and clorgyline inhibition was the same (50% recovery after 12-15 days), which indicates similar rate of synthesis of MAO in different cell types.

Selective inhibition of monoamine oxidase by p-aminosubstituted phenylalkylamines in catecholaminergic neurones.

The in vivo inhibition of monoamine oxidase (MAO) inside and outside noradrenergic and dopaminergic nerve terminals in the hypothalamus and striatum, respectively, was examined in the rat after oral administration of a series of substituted p-aminophenethylamines and some related compounds. This was achieved by measuring their ability to protect MAO from irreversible inhibition by phenelzine, determined by the deaminating activity of synaptosomal preparations in the absence and presence of maprotiline, a selective inhibitor of the uptake of noradrenaline, or of amfonelic acid, a potent inhibitor of the uptake of dopamine, with small (0.25 microM) concentrations of [14C]noradrenaline or [14C]dopamine as substrate. It was found that several of these compounds were much more potent in protecting MAO within the noradrenergic neurones than MAO in other cells. Since the inhibitors of the uptake of noradrenaline, desipramine and CPP 199 antagonized this preference for noradrenergic MAO it is concluded that these MAO inhibitors are accumulated in the noradrenergic neurones by the membranal uptake carrier. Hence the selectivity for MAO within noradrenergic neurones seems to reflect the ability of the compounds to be transported by this carrier. The structure-activity relationship obtained showed the greatest selectivity for the unsubstituted p-dimethylamino-(FLA 289), p-methylamino-(FLA 727) and p-amino-(FLA 334)-amphetamines, whereas the 2-fluoro compound (FLA 558) had the greatest potency. N,N-didesmethylamiflamine [FLA 668(+)] had an almost specific effect in the noradrenergic nerve terminals. The primary p-amino derivatives, FLA 334 and FLA 668, produced a marked selective protection of MAO in dopaminergic nerve terminals, whereas the tertiary and secondary derivatives had much less preference for dopaminergic MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of monoamine oxidase in 5-hydroxytryptaminergic neurones by substituted p-aminophenylalkylamines.

A series of substituted p-aminophenethylamines and some related compounds were examined with regards to the inhibition of monoamine oxidase (MAO) in vivo inside and outside 5-hydroxytryptaminergic neurones in the rat hypothalamus. This was recorded as the protection against the irreversible inhibition of MAO produced by phenelzine by determining the remaining deaminating activity in the absence and presence of citalopram using a low (0.1 microM) concentration of [14C]-5-hydroxytryptamine (5-HT) as substrate. Some of the phenethylamines were much more potent inside than outside the 5-hydroxytryptaminergic neurones. This neuronal selectivity was antagonized by pretreatment of the rats with norzimeldine, a 5-HT uptake inhibitor, which indicates that these compounds are accumulated in the 5-HT nerve terminals by the 5-HT pump. Selectivity was obtained for compounds with dimethyl, monomethyl or unsubstituted p-amino groups. An isopropyl group appears to substitute for the dimethylamino group but with considerably lower potency. Compounds with 2-substitution showed selectivity for aminergic neurones and this effect decreased with increased size of the substituent. The 2,6-dichloro derivative FLA 365 had, however, no neuronal selective action but was a potent MAO inhibitor. Substitutions in the 3- and 5-positions decreased both potency and selectivity. Prolongation of the side chain with one methylene group abolished the preference for the MAO in 5-hydroxytryptaminergic neurones although the MAO inhibitory potency remained. The selectivity disappeared by increasing the alpha-substituent to an ethyl group but remained for the alpha,alpha-dimethyl substituted derivatives. It is concluded that compounds which are (1) transported by the 5-HT pump and (2) potent reversible MAO-A inhibitors produce pronounced inhibition of MAO in 5-hydroxytryptaminergic neurones.

Effects of acute and repeated administration of amiflamine on monoamine oxidase inhibition in the rat.

The inhibitory effect on monoamine oxidase (MAO) of the reversible MAO-A inhibitor (+)-4-dimethylamino-2,alpha-dimethylphenethylamine [amiflamine, FLA 336(+)] was evaluated in the rat after acute and repeated (twice daily for two weeks) oral treatment. MAO activity was measured ex vivo in slices from the hypothalamus and the duodenum for both MAO-A and MAO-B. Amiflamine selectively inhibited the A form of MAO after repeated as well as after acute treatment (ED50 approximately 7 mumoles/kg both acute and repeated). In the brain slices this inhibition corresponded to a decrease in the concentration of 5-hydroxyindoleacetic acid (5-HIAA) and to an increase in the concentration of 5-HT in the hypothalamus, the hippocampus and the striatum. The concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) was decreased in the striatum to the same extent as the decrease in the 5-HIAA concentrations. The effect on the homovanillic acid (HVA) concentration was somewhat weaker as was the increase in the concentration of dopamine. No essential difference was found after acute and chronic treatment on the amine and metabolite levels. The MAO activity returned to normal 24 hours after final dosing. A large difference between the neuronal and the extraneuronal protection against the phenelzine-induced irreversible MAO inhibition in the hypothalamus was found after both acute and repeated treatment. The ED50 of the protection within the serotonergic neurons was 1.3 mumoles/kg p.o. (acute) and 0.75 mumoles/kg p.o. (repeated). Amiflamine was 3 times less potent within noradrenergic neurons than within serotonergic neurons. A brain to plasma ratio of about 20:1 was found for amiflamine and its metabolites. The plasma and the brain concentrations of the N-demethylated metabolite [FLA 788(+)] exceeded that of amiflamine after a single dose, whereas the N,N-demethylated [FLA 668(+)] was found in low concentrations. The effect on MAO-A correlated significantly with the plasma and the brain concentration of FLA 788(+).

Selective inhibition of monoamine oxidase in monoaminergic neurons in the rat brain.

The prevention by six reversible and selective monoamine oxidase-A (MAO-A) inhibitors (alpha-ethyl-tryptamine, harmaline, 4-methoxyamphetamine, amiflamine [FLA 336(+)], N-desmethylamiflamine [FLA 788(+)] and N,N-desmethylamiflamine [FLA 668(+)] of the phenelzine-induced irreversible MAO inhibition in the rat brain was examined. By using crude synaptosome preparations of hypothalamus and striatum incubated with low substrate concentrations of 14C-serotonin (1 X 10(-7) M), 14C-noradrenaline (2.5 X 10(-7) M) and 14C-dopamine (2.5 X 10(-7) M) in the absence and presence of selective amine uptake inhibitors (alaproclate, maprotiline and amfonelic acid, respectively), it was possible to determine the deaminating activities inside and outside the specific aminergic synaptosomes. Thus, with this technique the protection of MAO by the reversible inhibitors administered orally 1 h prior to the subcutaneous injection of phenelzine against the phenelzine effect could be determined inside and outside the specific aminergic neurons. It was found that alpha-ethyltryptamine, 4-methoxyamphetamine and particularly amiflamine and FLA 788(+) were more potent inside than outside the serotonergic neurons. FLA 668(+) was a selective inhibitor of noradrenergic MAO, to which also 4-methoxyamphetamine, amiflamine and FLA 788(+), but not alpha-ethyltryptamine had some preference. Harmaline had no certain preference for MAO in any of the aminergic neurons. At high doses of FLA 668(+) a preference for dopaminergic MAO was observed. Since pretreatment of the rats with norzimeldine or desipramine antagonized the preferences for serotonergic or noradrenergic MAO, it is plausible to conclude that the compounds showing these preferences are accumulated in the neurons by the membranal uptake systems.

Synthesis and monoamine oxidase inhibitory activities of alpha-allenic amines in vivo and in vitro. Different activities of two enantiomeric allenes.

A series of 15 alpha-allenic amines, including primary, secondary, and tertiary ones, was synthesized, partly by organocopper chemistry. Their ability to inhibit mouse and rat brain mitochondrial monoamine oxidase (MAO) in vivo and in vitro, respectively, was evaluated. Almost all compounds were quite potent inhibitors of MAO, some as potent as deprenyl. Like deprenyl, most of the compounds were selective inhibitors of the B form of MAO. the two enantiomeric forms of N-methyl-N-(2,3-pentadienyl)benzylamine (2) were prepared and the R-(-) form was found to be 2.7 times as active as the (+) form in vivo and 25 times as active in vitro. Most of the compounds were tested for their ability to potentiate the phenylethylamine (PEA) response in mice, and a good correlation between the potency of MAO inhibition and PEA potentiation was found. Compound 5, as the only compound tested, did not potentiate the blood pressure response to tyramine.