James A. Totaro

Fenfluramine: long-term reduction in brain serotonin (5-hydroxytryptamine).

A single oral dose of 15 mg/kg of fenfluramine reduced the level of serotonin in rat brain to 48, 51 and 63% of control at 1, 15 and 30 days, respectively, after administration. 3 mg/kg p.o. of the drug caused a smaller but significant diminution in brain serotonin. At the 3 mg/kg dose level, the decreases in serotonin were, at least partially, cumulative following multiple injections spaced 24 hr apart. Brains removed 14 days after the 5th daily injection of 5 mg/kg p.o. of fenfluramine had only 60% of the concentration of serotonin found in brains from control animals. These findings demonstrate that fenfluramine has a long-lasting action on serotonin-containing neurons in brain.

A 5‐HYDROXYTRYPTAMINE‐LIKE MODE OF ANORECTIC ACTION FOR 6‐CHLORO‐2‐[1‐PIPERAZINYL]‐PYRAZINE (MK‐212)

1 The mechanism of the reduction in food consumption elicited by 6‐chloro‐2‐[1‐piperazinyl]‐pyrazine (MK‐212) administered systemically was investigated in the rat. (+)‐Fenfluramine and (+)‐amphetamine were included in some studies for comparative purposes. 2 Pretreatment with methergoline, a 5‐hydroxytryptamine (5‐HT) antagonist, reduced the magnitude of the anorectic effect of 1.5 and 3 mg/kg of MK‐212, while the anti‐5‐HT agents, cyproheptadine and cinanserin, were likewise effective against the 3 mg/kg dose. 3 Xylamidine, an antagonist of 5‐HT that penetrates poorly into the central nervous system, completely blocked the decrease in food intake caused by 5‐HT administered peripherally, while not antagonizing an equianorectic dose of MK‐212. 4 Reduction of brain 5‐HT by intraventricular injection of 5,6‐dihydroxytryptamine, intraperitoneal administration of p‐chloroamphetamine or placement of a lesion in the region of the median raphe nucleus diminished the anorectic response to 3 mg/kg of MK‐212. The anorectic effect of amphetamine was reduced by p‐chloroamphetamine or lesion in the raphe, but not by 5,6‐dihydroxytryptamine. The decrease in food consumption produced by 1.5 mg/kg of MK‐212 was antagonized by prior treatment with p‐chloroamphetamine, but not by 5,6‐dihydroxytryptamine. 5 Haloperidol, which blocks receptors for dopamine, antagonized the anorexigenic effect of amphetamine, but was ineffective in offsetting the action of MK‐212, 3 mg/kg. 6 Pretreatment with chlorimipramine to inhibit the 5‐hydroxytryptaminergic uptake mechanism did not affect the anorectic response to 3 mg/kg of MK‐212, whereas the response to fenfluramine was diminished. 7 The results indicate that the anorectic action of MK‐212 involves a 5‐HT‐like component which is more evident at the higher dose level of the compound. The anorexigenic property of MK‐212 may depend, at least partly, upon the integrity of 5‐HT‐containing neurones in the central nervous system.

Interaction of taurine and β-alanine with central nervous system neurotransmitter receptors

Taurine, a weak β-adrenergic agonist in rat pineal cultures, causes a 32% increase in the binding of the β-adrenergic antagonist 3H-dihydroalprenolol to rat brain membranes at a concentration of 100 mM. No significant effect was seen at 10 mM however. The amino acid is also effective in displacing 3H-diazepam (Ki, 32.8 mM) and 3H-muscimol (Ki, 32.4 μM), ligands for the benzodiazephine and GABA receptor, respectively. β-Alanine, a taurine analog, is more effective in displacing 3H-diazepam (Ki, 13.8 mM) and 3H-muscimol (Ki, 9 μM) but shows no significant enhancement of 3H-dihydroalprenolol binding. While endogenous taurine levels in brain are in the millimolar range, the physiological significance of these observations remains to be determined.

Inhibition of the serotoninergic uptake system by MK-212 (6-chloro-2-[1-piperazinyl]-pyrazine)

Summary MK-212 inhibited the uptake of 3H-serotonin in cerebral cortical tissue with an EC50 of about 10 μM. At 10 μM, MK-212 did not affect the uptake of 3H-norepinephrine. Inhibition of serotonin and catecholamine uptake in vivo was assessed by determining the effect of pretreatment with MK-212 on the ability of p-chloroamphetamine and H 77/77 to decrease, respectively, the concentration of serotonin and catecholamines in rat brain. MK-212, 2.5–10 mg/kg i.p., produced a dose-related antagonism of the serotonin-lowering action of p-chloroamphetamine, while the H 77/77-induced reductions in norepinephrine and dopamine were not inhibited by prior administration of MK-212. Inhibition of the neuronal reuptake system for serotonin may contribute to the previously described serotoninmimetic action of MK-212 in the central nervous system.

Fenfluramine-induced enhancement of confinement motor activity: An indirect 5-hydroxytryptamine-like action?

Abstract Fenfluramine, administered to rats at anorectic dose levels, reduced open field activity and enhanced confinement motor activity, demonstrating the mixed depressant and stimulant characteristics of the compound. Studies designed to elucidate the mechanism of action for the confinement motor activity-enhancing effect of fenfluramine produced the following results: (1) confinement motor activity was also increased by giving MK-486 + 5-hydroxytryptophan, (2) methergoline and cinanserin, antagonists of 5-hydroxytryptamine, reduced the confinement motor activity-enhancing actions of fenfluramine and MK-486 + 5-hydroxytryptophan but not that of methylphenidate, (3) following p -chlorophenylalanine-induced reduction in brain monoamines (5-hydroxytryptamine: −73%, norepinephrine: −16%, dopamine: −9%), the action of fenfluramine was diminished, (4) 13 days after injecting 5,6-dihydroxytryptamine intraventricularly (5-hydroxytryptamine: −51%, norepinephrine: −9%, dopamine: −10%), the enhancement of confinenment motor activity elicited by fenfluramine or MK-486 + 5-hydroxytryptophan was augmented. It was concluded that that fenfluramine increases confinement motor activity via a 5-hydroxytryptamine-like action. That this action of fenfluramine was affected by both p -chlorophenylalanine and 5,6-dihydroxytryptamine indicates an interaction with central 5-hydroxytryptamine-containing neurones. Denervation supersentivity in 5,6-dihydroxytryptamine-treated animals may accout for the opposite nature of the effect produced by pretreatment with p -chlorophenylalanine and 5,6-dihydroxytryptamine.

Interaction of 2‐[β‐(4‐hydroxyphenyl)ethylaminomethyl]tetralone (BE‐2254: ‘HEAT’) with catecholamine receptors in rat brain membranes

2 [ /3 (4 Hydroxyphenyl) ethylaminomethyl] tetralone (BE-2254: ‘HEAT’) has been shown to block peripheral a-adrenoceptors (Benthe, Gothert & Tuchinda, 1972; Baumgarten, Gothert & others, 1972). Indirect pharmacological evidence indicates that BE-2254 administered systemically can also block central catecholamine receptors and impair catecholamine receptor-mediated responses (Clineschmidt, Pflueger & others, 1975a). In this latter study, BE-2254 was found to be approximately equipotent with chlorpromazine in blocking central noradrenaline receptors but was much less active than either chlorpromazine or haloperidol as a blocker of central dopamine receptors. Biochemical examination of the effects of BE-2254 on central catecholamine metabolism also indicated a preference of this compound for noradrenaline rather than dopamine receptors (Clineschmidt, Totaro & others, 1975b). These studies have now been extended to the in vitro level by examining the affinity of BE-2254 for central cc-, pand dopaminergic catecholamine receptors using the following binding assays: a-receptor; binding of [3Hj‘dihydroergocryptine (3H-DHE) (Greenberg & Snyder, 1977) and [3H](2-([2’,6’-dimethoxy]phenoxyethy1amino)methyl benzodioxan) (3H-WB-4101) (Greenberg, U’Prichard & Snyder, 1976) to rat cerebral cortical membranes; /3-receptor; binding ~f[~H]dihydroalprenolol (3H-DHA) to rat brain crude synaptosomal membranes (Alexander, Davis & Lefkowitz, 1975); dopamine receptor; binding of [3H]haloperidol (3HHAL) to rat caudate membranes (Burt, Creese & Snyder, 1976). The ligand concentrations used, total radioactivity bound (d min-l mg-’ original wet weight of tissue), total counts bound per assay tube and per cent specific binding were as follows~ 3H-DHE; 0.4 n M ; 128 d min-’; 1000 c min-’; 70%: 3H-WB-4101 ; 0.22 nM; 70 d min-‘; 530 c min-’; 75%: 3H-DHA; 18 nM; 168 d min-l; 1600 c min-l; 80%: 3H-HAL; 1 nM, 840 d min-’; 1600 c min-l; 50%. Counting efficiency for the 3Hligand/filter disk complexes in Amersham PCS Scintillation cocktail was 3840%.

Bradykinin agonist activity of a novel, potent oxytocin antagonist

From a series of potent cyclic hexapeptide oxytocin (OT) antagonists, a compound that exhibited significant bradykinin (BK) agonist activity was identified. L-366,811 (cyclo[L-proline-D-tryptophan-L-isoleucine-D-pipecolic acid-L-piperazine-2-carboxylic acid-N-Me-D-phenylalanine]) stimulated phosphatidylinositol (PI) turnover in rat uterine slices in vitro (approximately EC50, 2 microM) with a maximal effect (15-fold increase over basal) greater than that obtained for either BK or OT. L-366,811 also elicited dose-related contractions of the isolated rat uterus, producing measurable effects at 100 nM. Several other equally potent OT antagonists from the cyclic hexapeptide structural class were either less potent or inactive as activators of uterine PI turnover or contractility. The stimulatory effects of L-366,811 on uterine PI turnover and contractions were blocked by BK antagonists but not by an arginine vasopressin (AVP)/OT antagonist. In radioligand binding studies, L-366,811 exhibited moderate affinity (IC50, 360 nM) for the [3H]BK binding site in rat uterus, consistent with its potency in the functional models. These results indicate that L-366,811 exhibits BK agonist activity in rat uterus in vitro.

Increased concentrations of 3‐methoxy‐4‐hydroxyphenylethyleneglycol and homovanillic acid in rat brain after treatment with BE‐2254 (“HEAT”)

An accelerated turnover of catecholamines in the brain produced by neuroleptics and certain other drugs has been attributed to a compensatory increase in activity of catecholaminergic neurons in response to receptor blockade (Carlsson & Lindqvist, 1963; Laverty & Sharman, 1965; Da Prada & Pletscher, 1966; AndCn, Corrodi & others, 1967; AndCn, Butcher & others, 1970). The increased release and catabolism of the catecholamines leads in turn to higher brain concentrations of 3-methoxy-4hydroxyphenylethyleneglycol sulphate (MOPEG-SO,) or homovanillic acid (HVA), depending upon whether the receptors blocked are sensitive, respectively, to noradrenaline or to dopamine (AndCn, Roos & Werdinius, 1964; Laverty & Sharman, 1965; Bartholini, Haefely & others, 1972; Meek & Neff, 1973; Keller, Bartholini & Pletscher, 1973 ; Braestrup, 1974). We recently suggested that BE-2254, 2-[,8-(4hydroxypheny1)-ethylaminomethyll-tetralone (“HEAT”), blocks receptors in the brain for both noradrenaline and dopamine, the former at somewhat lower doses than the latter (Clineschmidt, Pflueger & others, 1975). The peripheral or-adrenoceptor blocking activity of BE-2254 had previously been shown by Benthe, Gothert & Tuchinda (1972). To gain further information about the central actions of BE-2254, we have examined its effect on MOPEG-SO, in hypothalamic tissue and HVA in striatal tissue, as well as on the whole brain concentrations of noradrenaline and dopamine. Female Charles River (CRCD COBS) rats, 16CL200 g, were decapitated 2.5 h after treatment (i.p.) with BE-2254 or vehicle (1 % methylcellulose). The animals to be used for subsequent determination of MOPEG-SO, or HVA were additionally treated 30 min later with probenecid (200 mg kg-l, i.p.), to block efflux of the metabolites from the brain. MOPEG-SO, in the hypothalamus was determined according to Meek & Neff (1972), and striatal HVA according to Juorio, Sharman & Trajkov (1966). Dissection of the brain was as described by Glowinski & Iversen (1966). Catecholamines were adsorbed from neutralized perchloric acid tissue extracts on alumina and eluted with 0.1 N HCI (Anton & Sayre, 1962), then assayed fluorometrically (Porter, Totaro & Burcin, 1965). MOPEG-SO, in hypothalamic tissue was elevated as a consequence of treatment with 1.25, 5 or 20 mg kg-l of BE-2254, whereas HVA in striatal tissue was signifi-