Thijs de Boer

Stimulation of D2-dopamine receptors in rat neostriatum inhibits the release of acetylcholine and dopamine but does not affect the release of γ-aminobutyric acid, glutamate or serotonin

D1-dopamine receptor stimulation did not affect the K+-induced release of [3H]GABA, [3H]glutamate, [3H]serotonin, [3H]dopamine and [14C]acetylcholine from slices of rat neostriatum. D2-dopamine receptor stimulation did not change the release of [3H]GABA, [3H]glutamate and [3H]serotonin, but inhibited the release of both [3H]dopamine and [14C]acetylcholine; this inhibition was antagonized by (-)-sulpiride. The release-inhibiting dopamine autoreceptors and the post synaptic dopamine receptors mediating the inhibition of acetylcholine release appear to be pharmacologically similar and can be classified as D2-receptors.

The α2-adrenoceptor antagonist Org 3770 enhances serotonin transmission in vivo

Abstract The antidepressant properties of Org 3770 (1,2,3,4,10,14b-hexahydro-2-methylpyrazinol[2,1-a]pyridol[2,3-c][2]benzazepine) may be mediated via blockade of auto and hetero α2-adrenoceptors. On-line microdialysis studies in freely moving rats showed Org 3770 (2 mg/kg s.c.) to increase hippocampal 5-HT (5-hydroxytryptamine) and dihydroxyphenyl acetic acid (DOPAC) — a measure of noradrenergic activity — by approximately 80%. The increase in DOPAC is probably caused by blockade of α2-autoreceptors on noradrenergic terminals. Indirect α1-adrenoceptor-mediated enhancement of 5-HT cell firing and direct blockade of inhibitory α2-heteroceptors located on 5-HT terminals are held responsible for the increase in extracellular 5-HT.

The Selective α2-Adrenoceptor Antagonist Mirtazapine (Org 3770) Enhances Noradrenergic and 5-HT1A-Mediated Serotonergic Neurotransmission

SummaryMirtazapine (Org 3770) is a selective antagonist at α2-adrenergic auto- and heteroreceptors, which are involved in regulation of neuronal noradrenaline (norepinephrine) and serotonin (5-hydroxytryptamine; 5-HT) release. It was identified as a potential antidepressant in rat sleep studies, showing characteristic REM sleep suppression, as well as activity in the bulbectomised rat and an operant behaviour model. However, mirtazapine does not inhibit monoamine reuptake and is inactive in classical tests predictive of antidepressant activity (antagonism of reserpine-induced hypothermia, Porsolt test, and muricidal behaviour). As an α2-antagonist, mirtazapine inhibits clonidine-induced mydriasis and resembles idazoxan in evoking conditioned taste aversion. In addition, mirtazapine induces lower lip retraction, a response characteristic of 5-HT1A receptor stimulation, and resembles the 5-HT1A agonist 8-hydroxy-dipropylaminotetraline (8-OH-DPAT) in producing conditioned taste aversion. Thus, mirtazapine may have indirect serotonin-enhancing effects, since its affinity for 5-HT1A receptors is low.As a consequence of noradrenergic facilitation, mirtazapine increases the firing of serotonergic raphe neurons and antagonises the inhibitory effects of noradrenaline on serotonergic terminals. In combination, these effects offer a mechanistic basis for the drug’s observed stimulatory effect on hippocampal serotonin release. Because mirtazapine blocks 5-HT2 and 5-HT3 receptors, 5-HT1-mediated transmission is selectively enhanced, as reflected in its 5-HT1A-like behavioural effects.In conclusion, noradrenergic activation via (α2-autoreceptor blockade and the consequent indirect enhancement of serotonergic transmission probably underlie the marked antidepressant activity of mirtazapine. Blockade of 5-HT2 and 5-HT3 receptors may account for the absence of those adverse effects associated with nonselective serotonergic activation and may also contribute to the anxiolytic and hypnotic properties of mirtazapine.

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

Synthesis and muscarinic activities of 3-(pyrazolyl)-1,2,5,6-tetrahydropyridine derivatives

A series of 3-(pyrazolyl)-1,2,5,6-tetrahydropyridine derivatives (B) was synthesized and tested for muscarinic activity in receptor binding assays using [3H]-oxotremorine-M (3H-OXO-M) and [3H]-pirenzepine (3H-PZ) as ligands. Potential muscarinic agonistic or antagonistic properties of the compounds were determined using binding studies measuring their potencies to inhibit the binding of 3H-OXO-M and 3H-PZ. Preferential inhibition of 3H-OXO-M binding was used as an indicator for potential muscarinic agonistic properties; this potential was confirmed in functional studies on isolated organs. All compounds with agonistic properties showed 3H-PZ/3H-OXO-M potency ratios in excess of 20. In contrast, for antagonists this ratio was found to be close to unity. Mono-halogenation resulted in compounds (4b and 4d) with M3 agonistic properties as shown by their atropine sensitive stimulant properties in the guinea pig ileum, but with very little or no M1 activity. Some minor in vivo effects were observed for both these compounds, with the iodinated compound 4d inducing salivation. Compound 4d also showed some positive mnemonic properties in rats where spatial short-term memory had been compromised by temporary cholinergic depletion. These data indicate that some M3 agonism may be desired in therapeutic agents aimed at the treatment of the cognitive deficits of Alzheimers disease patients.

ACTH/MSH-like peptides inhibit the binding of dopaminergic ligands to the dopamine D2 receptor in vitro

ACTH-(1-24) decreased the binding of the dopamine D2 receptor agonist, [3H]N-propylnorapomorphine ([3H]NPA), to rat striatal membranes in a concentration-dependent manner, with a Ki of 5 x 10(-7) M. Saturation curves for [3H]NPA binding in the presence of increasing concentrations of ACTH-(1-24) were performed. Scatchard analysis in the presence of ACTH-(1-24) revealed an increased dissociation constant (Kd), while the binding capacity (Bmax) was not affected by the peptide, suggesting an apparent competitive interaction between ACTH-(1-24) and [3H]NPA. ACTH-(1-24) also reduced the binding of the dopamine D2 receptor antagonist [3H]spiperone to striatal membranes, with a Ki of 10(-6) M. Much higher concentrations of ACTH-(1-24), up to 10(-4) M, were needed for the displacement of appropriate radiolabelled ligands from dopamine D1 receptors, serotonin 5-HT1A, serotonin 5-HT1B, muscarinic M1 acetylcholine and histamine H1 receptors. ACTH-(1-24) also inhibited the binding of [3H]spiperone to dopamine D2 receptors in membranes of the pituitary gland, the septum and the substantia nigra. ACTH-(1-39) and most ACTH fragments and analogs were less potent than ACTH-(1-24) in displacing [3H]NPA from the dopamine D2 receptor in striatal membranes. In general there was a relationship between displacing potency and chain length. ACTH-(7-16)-NH2 and benzyloxycarbonyl-ACTH-(8-16)-NH2, however, were more potent than ACTH-(1-24) in reducing the binding of [3H]NPA to dopamine D2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

cGMP formation and phosphoinositide turnover in rat brain slices are mediated by pharmacologically distinct muscarinic acetylcholine receptors.

The cGMP response and the accumulation of inositol monophosphate (IP) induced by carbachol were compared in slices of different rat brain structures. Basal cGMP and the responses of cGMP to carbachol appeared dependent on the concentration of added Ca2+, suggesting that distinct Ca(2+)-mediated and Ca(2+)-sensitive muscarinic receptor-mediated mechanisms stimulate guanylate cyclase. Regional responses of cGMP to carbachol or to direct stimulation of guanylate cyclase with sodium nitroprusside were markedly distinct, indicating that a major proportion of guanylate cyclase in the cortex, an intermediate proportion in other forebrain regions, and only a minor proportion in the brainstem is sensitive to muscarinic receptor stimulation. The regional patterns of IP and cGMP responses to carbachol were different in the forebrain. Maximal IP accumulation was found in the cortex, whereas cGMP responses were highest in the hippocampus. Moreover, IP and cGMP formation in the hippocampus were differently antagonized by atropine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), the M2-receptor subtype-preferring antagonist AF-DX 116 and the M1-selective antagonist pirenzepine. These data support the notion that the IP formation induced by carbachol in the forebrain predominantly is mediated by muscarinic receptors of the M1 subtype, and indicate the involvement of muscarinic receptors of the M3 subtype in the carbachol-induced cGMP formation.

Dopamine D1 and D2 receptors in the caudate nucleus of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats

A series of studies was carried out to characterize the binding properties of dopamine D1 and D2 receptors in membrane homogenates of the caudate nucleus of spontaneously hypertensive rats (SHR). Binding in SHR was studied at the age of 4 weeks when the rats were still in the prehypertensive phase, and at the age of 8 weeks, during the phase in which blood pressure is increasing dramatically; age-matched normotensive Wistar-Kyoto rats (WKY) were used as controls. Binding to dopamine D1 receptors was studied using [3H]SCH 23390. Antagonist binding of dopamine D2 receptors was performed with [3H]spiperone. At both ages no differences were found between SHR and WKY in affinity (Kd) or concentration (Bmax) of dopamine D1 and D2 receptors. Binding to the high affinity state of the dopamine D2 receptor was measured using the agonist [3H]N-n-propylnorapomorphine (NPA). No differences in Bmax or Kd were found between SHR and WKY at both ages studied, indicating that the ratio between dopamine D2 receptors in the high and in the low affinity state is not altered in spontaneous hypertension. Although the results do not reveal differences in affinities or concentrations of dopamine D1 or D2 receptors in the caudate nucleus between SHR and WKY, a role in the development of hypertension for the here described lack of receptor up-regulation in connection with our previous observation of lower release of dopamine in the caudate nucleus of SHR, cannot be excluded.

The effect of the potential antipsychotic ORG 5222 on local cerebral glucose utilization in freely moving rats

The effects of administration of different doses of the potential antipsychotic Org 5222 (0.01 and 0.1 mg/kg i.v.) upon local cerebral glucose utilization (LCGU) in 102 anatomically discrete brain regions of freely moving male Wistar rats were studied with the quantitative autoradiographic [14C]2-deoxyglucose technique. Glucose utilization was significantly changed after treatment with 0.01 and 0.1 mg/kg i.v. Org 5222 in two and four brain areas, respectively. Treatment with 0.01 mg/kg Org 5222 significantly reduced LCGU in the basal thalamus (the ventral posterior medial (VPM) and lateral (VPL) nuclei). After administration of 0.1 mg/kg Org 5222, significant reductions were seen in the basal thalamus (VPL and VPM) and the medio dorsal thalamic nuclei. A highly significant elevation in LCGU was observed in the lateral nucleus of the habenula. The results show that Org 5222 selectively reduced LCGU in thalamic structures and had no or minimal effect on limbic, cortical and nigrostriatal structures, suggesting that Org 5222 may have antipsychotic potential, without inducing cognitive and extrapyramidal side-effects.

Synthesis, and In vitro and In vivo muscarinic pharmacological properties of a series of 1,6-Dihydro-5-(4H)-pyrimidinone oximes

A series of 1,6-dihydro-5-(4H)-pyrimidinone oxime derivatives I was synthesized (Scheme 1, Tables 1 and 2) and tested for muscarinic activity (Table 3) in receptor binding assays using [3H]-oxotremorine-M (Oxo-M) and [3H]-pirenzepine (Pz) as ligands. Potential muscarinic agonistic or antagonistic properties of the compounds were determined using binding studies that measured their potencies to inhibit the binding of Oxo-M and Pz. Preferential inhibition of Oxo-M binding was used as an indicator for potential muscarinic agonistic properties; this potential was confirmed in functional studies on isolated organs. The series produced a wide range of active compounds with differing degrees of selectivity in M1, M2, and M3 functional models. Several compounds that have mixed agonist/antagonist profiles were able to reduce cholinergic-related cognitive impairments in models of mnemonic function. Substitutions (I, e.g. R2 or R3 = Me) at the 1,6-dihydro-5-(4H)pyrimidine ring disrupted binding and efficacy, whereas systematic variation of the oximes substituent R1 resulted in various degrees of potency and selectivity dependent on the nature of the substitution.