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ELSEVIER Neuroscience Letters 189 (1995)121-124 LETIERS

Interaction of fl-carbolines with central dopaminergic transmission in mice: structure-activity relationships

G i u s e p p e P i m p i n e l l a , M a u r a P a l m e r y *

Institute of Pharmacology and Pharmacognosy, University of Rome 'La Sapienza', P. le Aldo Moro, 5, 00185 Rome, Italy

Received 24 May 1994; revised version received 15 March 1995 ; accepted 15 March 1995

Abstract

Although several fl-carboline alkaloids display hallucinogenic properties in humans, their mechanism of action remains unclear. To ascertain their influence on central dopaminergic transmission, in this study we investigated the facilitating effect of low doses of various fl-carbolines on L-DOPA (250 mg/kg)-induced stereotypy in mice. Harmaline (0.075, 0.15 and 0.35 mg/kg) and harmine (0.15, 0.35 and 0.75 mg/kg) powerfully enhanced the degree of stereotypy, whereas 6-methoxy-harmalan, 6-methoxy-harman and harman were far less potent, augmenting stereotypy only at much higher doses (3 mg/kg). 6-Methoxy-tetrahydro-fl-carbolin (6-MeO-THBC) had only a weak effect at the dose of 3 mg/kg and tetrahydro-fl-carbolin (THBC) had no effect up to the dose of 3 mg/kg. The Ca 2+- channel blocker nimodipine (2.5 mg/kg) only slightly antagonized harmaline (0.15 mg/kg) facilitation of L-DOPA-induced stereotypy.

Keywords: fl-Carbolines; Harmine; Harmaline; Dopamine; L-DOPA; Stereotyped behavior

Harmine and harmztline, 7-methoxy-derivatives of the fl-carboline family, are psychotogenic in humans. Al- though 6-methoxy-harmalan, the 6-methoxy-derivative of 3,4-dihydro-fl-carboline, is also hallucinogenic in hu- mans, these effects have been attributed to an increased status of inspiration and introspection [1]. Otherwise, the hallucinogenic action of harman and 6-methoxy-harman has not yet been detected in man. Despite several attempts to relate their hallucinogenic properties to their effects on neurotransmitters, the mechanism of action of fl- carbolines remains unclear. The most important pharma- cological effects demonstrated for fl-carbolines are a re- versible inhibitory action on MAO-A [2] and antagonism on the benzodiazepine receptors [9]. However, other drugs that share these actions are not hallucinogenic.

Until now, most of the investigations conducted to as- certain the dopaminergic actions of fl-carbolines have reported that these compounds have antidopaminergic activity [5,18]. In addition, previous studies in our labora- tory have demonstrated that low doses of harmaline and other hallucinogenic drugs facilitate the contractile re- sponse to dopamine in isolated rabbit thoracic aorta

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[7,13,14] and that harmaline enhances dopamine-elicited hypertension in rats [8].

Swiss Albino CD-I mice of both sexes (Charles River, Italy), weighing 18-22 g, were housed 15-20 per cage in a light- (0800-1800 h) and temperature- (23 _ I°C) con- trolled room and kept in the laboratory for 3-4 days be- fore experiments. Each animal was used only once.

L-DOPA, 6-methoxy-harman, 6-methoxy-harmalan, 6- methoxy-tetrahydro-fl-carbolin (6-MeO-THBC) and tet- rahydro-fl-carbolin (THBC) were soluted by adding the stoichiometric quantity of 1 N HC1 and diluted with dis- tilled water. The doses refer to free bases. Harmine, har- man and harmaline hydrochloride were used and the doses refer to the salts. Nimodipine was soluted in 20% ethanol. Each treatment group consisted of at least 6 mice; control groups received vehicle. The injection vol- ume was 10 ml/kg i.p. or s.c. All drugs were purchased from Sigma Chem. Co.

L-DOPA was administered i.p. at the dose of 250 mg/kg and the tested fl-carbolines were injected s.c., at various doses, 10 min before L-DOPA. The mice were housed in groups of two animals in Plexiglass cages with sawdust on the bottom and their behavior was observed every 5 min for 1 h. The behavioral effects were assessed

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122 G. Pimpinella, M. Palmery / Neuroscience Letters 189 (1995) 121-124

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Fig. 1. Facilitating effects (mean rating score _+ SE) of the administra- tion of harmaline (0.075, 0.15 and 0.35 mg/kg s.c.) and of harmine (0.15, 0.35 and 0.75 mg/kg s.c.) on L-DOPA (250 mg/kg i.p.)-induced stereotypy in mice. **P < 0.0l versus controls.

according to the scoring system of Everett [4]. In brief, scores were assigned as follows: 1, unnatural position; 2, as before with occasional gnawing, jumping or opisto- tones; 3, slight increase in motor activity with occasional gnawing, jumping or opistotones; 4, marked increase in motor activity with frequent gnawing, jumping or opisto- tones; 5, as before but more marked; and 6, frenetic gnawing alternating with marked increase in motor activ- ity.

Results are reported as mean values _+ SE of the scores attributed during the observation hour. Data were ana- lyzed for statistical significance with the Kruskall-Wallis test (non-parametric ANOVA). Differences were consid- ered significant at P < 0.05.

At doses of 0.075, 0.15, and 0.35 mg/kg, harmaline enhanced L-DOPA-induced stereotypy in mice. The dose- effect curve had a steep slope (Fig. 1). The lowest dose already induced a significant effect which neither of the higher doses increased. Even higher doses, 0.75 and 1.5 mg/kg, increased the degree of stereotypy further, but caused convulsions and prostration, followed by death within 24 h.

6-Methoxy-harmalan had a much less potent facili- tating effect than harmaline (Fig. 1). Only the highest dose (3 mg/kg) induced a degree of stereotypy compara- ble to that elicited by harmaline. Over the dose range tested (0.15, 0.35 and 0.75 mg/kg) harmine had a signifi- cant dose-dependent effect (Fig. 2). A higher dose (1.5 mg/kg) increased the degree of stereotypy even further (results not shown). At all doses tested (0.75, 1.5 and 3 mg/kg) 6-methoxy-harman increased L-DOPA- induced stereotypy. Responses were not dose-related (Fig. 2).

Over the dose range tested (0.45, 1.5 and 3 mg/kg) harman and 6-MeO-THBC had weak effects on L-DOPA- induced stereotypy in mice and only the highest dose pro- duced a statistically significant action. THBC had no fa- cilitating effect up to the dose of 3 mg/kg.

The Ca2÷-channel blocker nimodipine, administered at the doses of 0.65, 1.5 and 2.5 mg/kg, only slightly an- tagonized the facilitating effect of harmaline (0.15 mg/kg) on L-DOPA-induced stereotypy and did so only at the highest dose (mean score _+ SE, 2.4 _+ 0.15 for the ni- modipine-treated group versus 3.0 +_ 0.09 for the control group; P < 0.05).

The primary finding of our study is that fl-carbolines dramatically facilitate dopaminergic transmission in the CNS. This contrasts with evidence from others showing that harmala alkaloids have antidopaminergic actions [5,17]. On the other hand it confirms evidence that har- maline facilitates dopamine-induced contractions in rabbit isolated aorta [7] as well as the hypertensive effect of dopamine in rats [8]. It also concurs with previous studies showing that harmine and harmaline administered in combination with L-DOPA or apomorphine cause violent 'jumping behavior' in rats [6,16].

Our findings provide evidence that in this experimental model the methoxy group in position 7 of the fl-carboline system is responsible for increasing the compound's po- tency. This explains why the two 7-methoxy-fl-carbo- lines, harmine and harmaline, are the two most potent compounds. In contrast, the aromaticity of the molecule seems to cause the opposite effect, so that harmine, which is aromatic, is less active than harmaline, which is not aromatic. For the same reason, 6-methoxy harman is less

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Fig. 2. Facilitating effects (mean rating score _+ SE) of the administra- tion of 6-methoxy-harman (6-MeO-harman 0.75, 0.15 and 3 mg/kg s.c.) and of 6-methoxy-harmalan (6-MeO-harmalan 0.75, 0.15 and 3 mg/kg s.c.), on L-DOPA (250mg/kg i.p.)-induced stereotypy in mice. *P < 0.05 versus controls; **P < 0.01 versus controls.

G. Pimpinella, M. Palmery / Neuroscience Letters 189 (1995) 121-124 123

potent than 6-methoxy harmalan. Moving the methoxy group to posit ion 6 decreases activity significantly, hence the great difference in potency between harmaline and 6- methoxy harmalan, and between harmine and 6-methoxy harman.

Because tetrahydro-~-carbolines are much less active than unsaturated compounds, the loss of the double bond in position 1 of the fl-carboline ring decreases potency still further. The methoxy group in position 6 apparently has a weak effect on activity, because 6-methoxy harman is only slightly more potent than harman itself and 6- MeO-THBC is only ,;lightly more potent than THBC (P < 0.05).

Our findings make it unlikely that the mechanism of action of fl-carbolines, in this experimental model, in- volves benzodiazepine receptors. Previous data show that harmaline, the most potent compound, has a K i for ben- zodiazepine receptors ~!hat exceeds 100 ktM [3], whereas harmaline given at a ,Jose of 10 mg/kg produces much lower brain concentralions [19]. This seems to exclude the possibil i ty that the harmaline doses used in our ex- periments acted on the GABAergic system.

Previous studies h~tve demonstrated that the GABA agonist muscimol facilitates amphetamine-induced stereo- typy in rats ]12], and others have reported that harmine given at high doses eahances, whereas G A B A agonists and antagonists suppress, apomorphine-induced jumping behavior in rats. T!~ese findings suggest that the GABAerg ic system is only partly involved [6].

It also seems unlikely that the observed effects could depend on the facilitating action off l -carbol ines on sero- toninergic transmission, because pretreatment with 5- hydroxy-tryptophan (HTP) antagonizes dopaminergic stereotyped behavior [17]. Moreover, binding studies have shown that harmaline interacts poorly with D1 and D2 dopaminergic receptors (and also with 5-HT, adrena- line, histamine, acetybzholine and opioid receptors ) [3], for this reason also excluding a direct receptor action.

/3-Carbolines are not likely to affect catecholamine uptake in vivo, because they are effective in vivo only at very high doses [ l 0].

The role of Ca 2÷ is unclear. Harmaline activates cal- cium channels in bulbar olives, and this action is antago- nized by the calcium channel-blocker 2-octanol [15], whereas nifedipine inhibits harmaline-induced facilitation of the contractile response to dopamine in rabbit isolated thoracic aorta [7]. Nimodipine is only slightly active in inhibiting L-DOPA-induced stereotypy, however, we have already shown that nifedipine, up to the dose of 5 0 # g / k g i.v.(higher doses caused marked hypotension), does not inhibit harmaline (400ktg/kg i.v.) facilitation of the dopamine (50 ktg/kg i.v.) hypertensive effect in anaes- thetized rats (unpublished data). Hence, even if Ca 2+ does intervene, it can be no more than a secondary mechanism.

In our opinion, a more likely explanation, supported also by the fact that harmaline influences Na+-dependent

transport mechanisms [3], is that it increases dopamine release in the corpus striatum, a structure known to be involved in stereotyped behavior. Dopamine release might also account for the beneficial effects of harmine in patients with Parkinson 's disease [11].

In conclusion, fl-carboline alkaloids facilitate dopa- minergic transmission in the CNS, probably by interact- ing with Na+-dependent membrane transporting systems.

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124 G. Pimpinella, M. Palmery / Neuroscience Letters 189 (1995) 121-124

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