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.

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1 A series of substituted p-aminophenethylamines and some related compounds were examined with regards to the inhibition ofmonoamine 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 yIM) concentration of ['4CJ-5-hydroxytryptamine (5-HT) as substrate. 2 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. 3 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. 4 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 a-substituent to an ethyl group but remained for the oco-dimethyl substituted derivatives. 5 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.

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 ['4C]5-hydroxytryptamine ([14Cj5-HT; 0.1 p M ) , [14C]noradrenaline (0.25 p M ) , [14C]3,4-dihydroxyphenylethylarnine ([I4C]dopamine; 0.25 p M ) , and [I4C]tyramine (0.25 p M ) was hindered by selective uptake inhibitors (citalopram, maprotiline, and amfonelic acid) in the incubation media. Thus, the difference between the deamination of I4C-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 M A 0 activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxins N-chloroethyl-N-ethy1-2-Address correspondence and reprint requests to Dr. S. B . amine; DOPEG, 3,4-dihydroxyphenylglycol; DSP4, N-chlocepted February 16, 1986. roethyl-N-ethyl-2-bromobenzylamine hydrochloride; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT or serotonin, 5-hydroxytryp-Ross at Research Laboratories, Astra Lakemedel AB, ,5151 85 t a m i n e ; M A O , m o n o a m i n e o x i d a s e ; 6 -O H D A , 6 -Sodertalje, Sweden. hydroxydopamine hydrochloride; PCA, p-chloroamphetamine Abbreviations used: dopamine, 3,4-dihydroxyphenylethyl-hydrochloride. U S A 79, 6385-6389.

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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.

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.

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