Peter A. Baumann

Blockade of presynaptic ?-receptors and of amine uptake in the rat brain by the antidepressant mianserine

SummaryMianserine (Org GB 94, Tolvon®) is 1, 2, 3, 4, 10, 14b-hexahydro-2-methyl-dibenzo [c, f] pyrazino [1, 3-a] azepine hydrochloride, a new antidepressant drug. Its effect on noradrenaline release and its capacity to inhibit amine uptake were investigated. Mianserine increased the release of 3H-noradrenaline from field-stimulated cortical slices previously labelled with the tritiated transmitter. The assumption that this effect is due primarily to the blockade of the presynaptic noradrenergic α-receptors is supported by the fact that mianserine failed to augment 3H-noradrenaline release further after blockade of the presynaptic α-receptors by phentolamine. In the reciprocal experiment, phentolamine failed to augment 3H-noradrenaline release after exposure of the slices to mianserine. The hypothesis is further reinforced by the fact that mianserine antagonized the reduction of 3H-noradrenaline release by clonidine in the same manner as the α-blocking drugs phentolamine and phenoxybenzamine. Mianserine inhibited noradrenaline uptake in vitro and in vivo (in the rat heart and midbrain-diencephalon synaptosomes from pretreated rats.) Only a marginal inhibition of serotonin uptake was observed.It therefore appears that mianserine increases the concentration of noradrenaline in the synaptic cleft by blocking the presynaptic α-receptors and inhibiting uptake. Whether or not this increase has functional consequences at postsynaptic noradrenergic receptor sites is unknown. It is possible, however, that postsynaptic receptor blockade counteracts the increase in available noradrenaline.

Depression of exploratory activity by clonidine in rats as a model for the detection of relative pre- and postsynaptic central noradrenergic receptor selectivity of α-adrenolytic drugs

SummaryThe effects of various α-adrenoceptorblocking drugs on the depression of exploratory activity (ambulation and rearing) induced by 0.1 mg/kg i.p. clonidine were investigated in the rat. In parallel experiments, the effects of the same drugs on pre- and postsynaptic α-receptors were determined in vitro (field-stimulated cortex slices and isolated vas deferens of the rat, respectively). Tolazoline, esproquine, yohimbine and piperoxan distinctly antagonized the inhibition of exploration produced by clonidine. All these drugs were found to possess relatively higher selectivity for the presynaptic α-receptors, as judged by the ratios of the concentrations inducing a 50% increase in field-stimulated 3H-noradrenaline-overflow and the concentrations required to shift the EC50 for the antagonism of noradrenaline-induced contractions of the vas deferens to the right by a factor of 2 (pA2, ratio <1): In contrast, phentolamine and phenoxybenzamine which showed preferential postsynaptic α-receptor blocking activity (ratio>1), potentiated rather than antagonized the effects of clonidine. Mianserin, although preferentially blocking the postsynaptic receptors, had no effect on clonidine-induced hypoactivity up to the high dose of 100 mg/kg i.p., probably because of its additional NA-uptake-inhibiting properties. The antagonism of clonidine by the selective presynaptic α-receptor blockers was observed within a limited dose-range. Increasing the doses above an optimal level, which varied from one compound to another, resulted in a decrease in the effect. It is suggested that this phenomenon reflects the counter-balancing postsynaptic α-adrenoceptor blockade occuring at higher concentrations of these drugs. In general, the results show a fairly good correlation between antagonism of clonidine in vivo and preferential blockade of presynaptic α-receptors in vitro. Clonidine-induced suppression of exploration therefore seems to be a valuable model for the investigation of drug interactions with α-adrenergic receptors in the central nervous system.

Oxaprotiline, a noradrenaline uptake inhibitor with an active and an inactive enantiomer

Abstract The new antidepressant drug, oxaprotiline, and its two enantiomers were investigated with respect to their effects on noradrenaline (NA) and serotonin (5-HT) uptake in vitro and in vivo after acute and repeated treatment. Moreover, the alpha-adrenolytic effects in vitro were also studied. Oxaprotiline proved to be a highly potent and selective inhibitor of NA uptake in rat synaptosomal preparations in vitro and in the rat heart and brain in vivo The NA uptake-inhibiting properties were found to be confined entirely to the (+)- or S-enantiomer: (−)- or R-oxaprotiline, the absolute configuration of which corresponds to that of the naturally occurring (−)-NA, was about 1000 times less potent than the (+)-form in vitro , and was inactive in vivo at doses exceeding the ED 50 of the latter 100-fold. The selectivity of oxaprotiline with respect to NA uptake inhibition was retained after 10 daily administrations. No sign of cumulation or attenuation of the effect was evident. No uptake-inhibiting effect of (−)-oxaprotiline appeared after 10 daily administrations of high doses, indicating that no racemization occurred in the organism. The α 1 -adrenoceptor antagonistic effect of oxaprotiline, as determined by the ability to displace [ 3 H]prazosin, was in the range of that of imipramine, the (−)-enantiomer being somewhat more potent than the (+)-form. In contrast to imipramine, oxaprotiline was devoid of α 2 -adrenoceptor antagonistic effects, as judged by the ability to affect the impulse-related release of [ 3 H]NA from rat cortical slices. Since oxaprotiline proved to be an effective antidepressant, clinical testing of its two enantiomers might be helpful with respect to the validation of the catecholamine hypothesis of depression. Moreover, in animal studies, they might help to determine which effects of antidepressants are related to NA uptake inhibition and which are not.

GABA and glutamate release affected by GABAB receptor antagonists with similar potency : no evidence for pharmacologically different presynaptic receptors

1 The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared. 2 The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50s) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348). 3 Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofens effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4 Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5 In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

Further evidence for negative feedback control of serotonin release in the central nervous system.

SummaryIn the presence of the serotonin (5-HT) uptake inhibitor citalopram (2×10−5 M), 5-HT, added to the superfusion medium, diminished the electrically induced 3H-overflow from rat cortex slices prelabelled with 3H-5-HT. A 50% decrease of the 3H-overflow was obtained with 6×10−7 M 5-HT. Of the 5-HT antagonists tested (methysergide, methergoline, methiothepin, cyproheptadine, cinanserin, mianserin, pizotifen), methiothepin (50% at 4.5×10−7 M), methergoline (31% at 10−6 M) and cinanserin (30% at 10−6 M) increased the stimulation-induced 3H-overflow. In some experiments the overflow of endogenous 5-HT was measured simultancously with 3H-overflow. The results were qualitatively and quantitatively very similar, indicating that 3H-overflow can be taken as a reliable measure of 5-HT release. The existence of a functional negative feedback control of 5-HT release mediated by presynaptic autoreceptors was confirmed by analysis of the 3H-overflow per pulse at stimulation frequencies between 0.1 and 50 pulses per second (pps). The 3H-overflow per pulse decreased with increasing stimulus frequency, reaching a low level at 1.6 pps corresponding to 30% of the initial value at 0.1 pps. Methiothepin did not increase the stimulation-induced 3H-overflow at 0.1 pps. The stimulation-induced 5H-overflow at 0.1 pps in the absence of methiothepin was equivalent to that at 1.6 pps in the presence of methiothepin. These results suggest that, under the given experimental conditions, at a stimulation frequency of 0.1 pps the 5-HT concentration in the synaptic cleft drops to very low levels, barely sufficient to induce negative feedback. Among a variety of drugs tested, LSD, dimethyltryptamine, m-chlorophenylpiperazine and clonidine decreased the stimulation-induced 3H-overflow, whereas salbutamol, propranolol, oxprenolol, spiroperidol and haloperidol had no effect. Quipazine, known as a 5-HT agonist, unexpectedly augmented the stimulation-induced 3H-overflow. This effect might be attributable partly to its 5-HT uptake inhibiting component, and also to blockade of presynaptic 5-HT receptors.

Potential involvement of a baclofen-sensitive autoreceptor in the modulation of the release of endogenous GABA from rat brain slices in vitro

SummaryThe effects of the GABAA agonist, muscimol, and of the enantiomers of the GABAB agonist, baclofen, on the release of endogenous GABA from slices of the rat cerebral cortex, striatum and hippocampus were measured by means of a HPLC method with electrochemical detection. Moreover, the effect of the GABAA antagonist, bicuculline, and of the frequency of stimulation were studied in cortical slices. The amount of endogenous GABA released per impulse from cortical slices decreased by about 50% when the frequency was increased from 0.25 Hz to 1 Hz. This might indicate that GABA inhibited its own release. (−)-Baclofen at 1 and 10 μM, but not its (+)-enantiomer, markedly inhibited the release of endogenous GABA, to a similar extent in all 3 areas investigated. The effect of (−)-baclofen was dependent on the frequency of stimulation: at lower frequencies (0.25 and 0.5 Hz) it was more marked than at a higher one (4 Hz). This would be expected from the results showing that the release of endogenous GABA decreases with increasing frequency, which suggests that this amino acid inhibits its own release. Muscimol at 10 μM, on the other hand, was ineffective in all 3 areas at a stimulation frequency of 0.5 Hz. Bicuculline (10 μM) at 4 Hz, at which autosuppression of GABA release is maximal did not enhance the release of endogenous GABA from cortical slices. With cerebellar or nigral slices, no adequate stimulation-induced release of endogenous GABA could be obtained under comparable conditions. These data are compatible with, but do not prove the existence of GABAB-type presynaptic autoreceptors modulating the release of this amino acid. More definite conclusions may possibly be drawn when a GABAB antagonist becomes available, which is expected to enhance GABA release under appropriate conditions.

Investigations on GABAB receptor-mediated autoinhibition of GABA release

SummaryIn this study, we have investigated the effects of phaclofen on the [3H] overflow from [3H]GABA prelabelled rat cortical slices and its interaction with the effects of (−)-baclofen in dependence of the stimulation frequency. (−)-Baclofen strongly depressed the [3H] overflow in the frequency range of 0.125 to 4 Hz to a constant residual level (ICIn50 = 0.37 μmol/l at 0.125 Hz), but became inactive above. The potency of the (+)-enantiomer was considerably weaker by a factor of nearly 1000. The GABAB antagonist, phaclofen, increased [3H] overflow at 300 μmol/l and, moremarkedly, at 3 and 1 mmol/l, respectively. However, the increase was virtually independent of the frequency between 0.125 and 16 Hz. If the compound interacted only with the putative GABAB autoreceptor involved in the regulation of GABA release, the extent of the enhancing effect should increase with increasing frequency because of the concomitant rise in synaptic GABA concentration. In order to further investigate this phenomenon, the IC50 of (−)-baclofen and antagonism of phaclofen against (−)-baclofen were determined at 0.125 Hz and 2 Hz, respectively. Whereas the IC50 of (−)-baclofen was 0.63 ± 0.04 μmol/l at 0.125 Hz, it increased to 4.88 + 0.45 μmol/l at 2 Hz. The pA10-values of phaclofen were about the same at both frequencies, whereas the pA2-values differed by a factor of 2.3. Therefore, the possibility should be considered that (−)-baclofen does not only interact with presynaptic GABA autoreceptors, but also may interact with other - presumably somatodendritic- GABAB-receptors whose pharmacology is not identical with that of the receptors by which (−)-baclofen exerts its effects on GABA release.

Effects of CGP 11305 A, a new reversible and selective inhibitor of MAO A, on biogenic amine levels and metabolism in the rat brain

SummaryCGP 11305A [4-(5-methoxy-7-bromo-benzofuranyl-2-)piperidine HCl], a reversible, selective inhibitor of MAO A, increased the levels of rat brain noradrenaline, dopamine, and serotonin dose-dependently and to approximately the same extent. Concomitantly, it lowered the levels of their metabolites, MHPG-SO4, HVA, DOPAC, and 5-HIAA. When compared with the irreversible MAO A inhibitor clorgyline, the effects of CGP 11305 A were of much shorter duration. Moreover, the increases of noradrenaline and serotonin and the decreases of their metabolites MHPG-SO4 and 5-HIAA were smaller after CGP 11305 A than after clorgyline in equieffective doses for MAO A inhibition. CGP 11305A, decreased the synthesis of catecholamines and serotonin less markedly than clorgyline. This is probably due to the reversibility of the interaction of the compound with MAO A. In contrast to CGP 11305 A, clorgyline increased the level of dopamine less than those of noradrenaline and serotonin. This is explained by assuming that dopamine synthesis is particularly sensitive to end product inhibition.CGP 11305 A also exhibited some inhibitory properties on the uptake of serotonin and, to a lesser degree, of noradrenaline in vitro and in vivo. Compared with MAO inhibition, however, uptake inhibition required 30–100 times higher doses.

CGP 28014, a new inhibitor of cerebral catechol-O-methylation with a non-catechol structure

SummaryCGP 28014 (N-(2-pyridone-6-yl)-N′,N′-di-n-propylformamidine) or its methanesulfonate salt CGP 28014 A was suspected to be a catechol-O-methyl-transferase (COMT) inhibitor because it was found to reduce the levels of homovanillic acid (HVA) and to increase those of 3,4-dihydroxyphenylacetic acid (DOPAC) in the rat striatum, after oral or intraperitoneal administration. These effects were maintained after repeated administration. The compound was only weakly active as a COMT inhibitor in vitro. However, its effect on striatal HVA and DOPAC was not prevented by pretreatment with the inhibitor of microsomal drug metabolizing enzymes in the liver, proadifen, indicating that, if CGP 28014 acts as a prodrug, its conversion to the active compound is not by oxidative metabolism in the liver. Also, there was no evidence that conversion to 2-amino-6-hydroxypyridine could explain its effects. The in vivo effect of CGP 28014 was substantiated in two additional in vivo test systems. Thus, it inhibited the accumulation of 3-methoxytyramine in the rat striatum after MAO inhibition by clorgyline, and the formation of O-methyl-DOPA from exogenously administered DOPA. It proved to be equipotent or nearly so with tropolone, and also showed a similar duration of action. Similar to tropolone, it increased S-adenosylmethionine levels in the striatum. Pyrogallol, on the other hand, decreased them, because being a substrate of COMT, it consumes methyl groups. This suggests that CGP 28014 does not inhibit COMT because it is a substrate of the enzyme.No effects were noted on catecholamine and serotonin concentrations in rat brain; no effects on noradrenaline uptake in rat heart, or on serotonin uptake in rat brain at doses of 30 mg/kg i.p. and above were found. Slight increases of brain tryptophan and 5-hydroxyindoleacetic acid, found occasionally, may indicate a minimal enhancing effect on serotonin turnover.In receptor binding tests, CGP 28014 at 10−5 mol/l showed very weak or no interactions at all with αl-, α2-and ß-noradrenergic, 5-HT1A, 5-HT1B, 5-HT2, muscarinic cholinergic, histamine, H1 and H2, GABAA, benzodiazepine, adenosine A1 and A2, δ-, µ- and κ-opiate, and substance P receptors in vitro, and D2 and 5-HT2 receptors in vivo. The compound holds a potential to improve the efficacy of L-DOPA in the treatment of parkinsonism.

Selective inhibition of noradrenaline and serotonin uptake by C 49802-B-Ba and CGP 6085 A.

The effects of two new compounds, 1-(1-methylamino-2-hydroxy-3-propyl)-dibenzo[b,e]bicyclo[2,2,2]octadiene-HCl (C 49802-B-Ba) and 4-(5,6-dimethyl-2-benzofuranyl) piperidine HCl (CGP 6085 A), on noradrenaline (NA) and serotonin (5-HT) uptake were investigated in different test systems, CGP 6085 A is a very potent and selective inhibitor of 5-HT uptake in rat brain (ED50 1-4 mg/kg p.o., depending on test system). Doses up to 1000 mg/kg p.o. did not inhibit NA uptake. C 49802-B-Ba is a potent and selective inhibitor of NA uptake in rat brain (ED50 5-10 mg/kg p.o. depending on test system) and heart (ED50 1.5 mg/kg p.o.). At 300 mg/kg p.o., this compound caused no inhibition of 5-HT uptake.