5-Hydroxytryptamine induced excitation and inhibition in the subthalamic nucleus:action at 5-HT2C, 5-HT4 and 5-HT1A receptors

Extracellular single-unit recordings in mouse brain slices were used to determine the effect of exogenously applied 5-HT on STN neurones. Recordings were made from 74 STN cells which fired action potentials at a regular rate of 7.19 ± 0.5 Hz. In 61 cells (82%), 5-HT application increased STN neurone firing rate (10 μM, 180 ± 16.8%, n = 35) with an estimated EC 50 of 5.4 μM. The non-specific 5-HT2 receptor agonist α-methyl 5-HT (1-10 μM) mimicked 5-HT induced excitations (15 cells). These excitations were significantly reduced by pre-perfusion with the specific 5-HT2C receptor antagonist RS102221 (500 nM, 9 cells) and the 5HT4 antagonist GR113808 (500 nM, 7 cells). In 6 cells (8%) 5-HT induced biphasic responses where excitation was followed by inhibition, while in 7 cells (9%) inhibition of firing rate was observed alone. Inhibitory responses were reduced by the 5-HT1A antagonist WAY100135 (1 μM, 4 cells). No inhibitory responses were observed following α-methyl 5-HT applications. Both the excitations and inhibitions were unaffected by picrotoxin (50 μM, n = 5) and CNQX (10 μM, n = 5) indicative of direct postsynaptic effects. Thus, in STN neurones, 5-HT elicits two distinct effects, at times on the same neurone, the first being an excitation which is mediated by 5-HT 2C and 5-HT4 receptors and the second an inhibition which is mediated by 5-HT1A receptors. © 2005 Elsevier Ltd. All rights reserved.

Cholecystokinin-1 receptor antagonists:5-hydroxy-5-aryl-pyrrol-2-ones as anticancer agents

A new class of 5-arylated 5-hydroxypyrrolones was derived from mucochloric acid in 2 synthetic steps and the chemical structure was confirmed additionally by X-ray analysis. Using a radiolabelled binding assay, potent CCK1 selective ligands were identified (CCK1: 12 nM) and the antagonism was confirmed by using isolated tissue preparations. A series of isobutyl derivatives displayed unsurmountable CCK antagonistic properties and in vitro excellent inhibition of proliferation was obtained in cholecystokinin related cancer cell lines in the nanomolar range. Finally, using xenograft studies in nude mice, two selected pyrrolone derivatives, X = H and X = F a fluorinated analogue (PNB-028), showed a strong inhibition of tumour growth in a chemo-resistant colon cancer-(MAC 16) and a human pancreatic cell line (MIAPACA) at 50 mg kg-1 by oral administration.

Aspects of tic like behaviour and serotonergic control

Tic-like movements in rodents bear close similarities to those observed in humans both pharmacologically and morphologically. Pharmacologically, tics are modulated by serotonergic and dopaminergic systems and abnormalities of these systems have been reported in Tourette's Syndrome (TS). Therefore, serotonergic and dopaminergic modulation of tics induced by a thyrotrophin-releasing hormone (TRH) analogue were studied as possible models for TS. The TRH analogue MK771 induced a variety of tic like movements in mice; blinking fore-paw-licking and fore-paw-tremor were quantified and serotonergic and dopaminergic modulation was investigated. The selective dopamine D1 receptor antagonists SCH23390 and SCH39166 and dopamine D2 antagonists raclopride and sulpiride had no effect on MK771 induced blinking. The D1 antagonists attenuated fore-paw-tremor and -licking while the D2 antagonists were generally without effect on these behaviours. Ketanserin (5-HT2A/ alpha-1 antagonist) and ritanserin (5-HT2A/2C antagonist) were able to attenuate MK771-induced blinking and ketanserin, mianserin (5-HT2A/2C antagonist) and prazosin (alpha-1 adrenoceptor antagonist) were able to attenuate MK771-induced fore-paw-tremor and -licking. The 5-HT2C/2B antagonist SB200646A was without effect on blinking and fore-paw-licking but dose-dependently potentiated fore-paw-tremor. The 5-HT1A agonists 8-OH DPAT and buspirone attenuated blinking at the lower doses tested but were ineffective at the higher doses; the converse was found for fore-paw-licking and -tremor behaviours.The effects of these ligands appeared to be at a postsynaptic 5-HTlA site since para-chlorophenylalanine was without effect on the manipulation of these behaviours. (S)-W A Y100135 was without effect on MK771-induced behaviours, spontaneous and DOl-induced head shakes. Because kynurenine potentiates head shakes and plasma concentrations are raised in TS patients the effects of kynurenine on the 5-HT2A/2C agonist DOl mediated head shake were established. Kynurenine potentiated the DOl head shake. Attempts were made to correlate serotonergic unit activity with tic like behaviour in cats but this proved unsuccessful. However, the pharmacological understanding of 5-HTlA receptor function has been hampered because of the lack of selective antagonists for this site. For this reason the effects of the novel 5-HTlA antagonists (S)-WA Y- 100135 and WAY -100635 were tested on 5-HT single-unit activity recorded from the dorsal-raphe-nucleus in the behaving cat. Both drugs antagonised the suppression of unit activity caused by 8-0H DPAT. (S)-WA Y-100135 reduced unit activity whereas WAY-100635 increased it. This suggests that WAY-100635 is acting as an antagonist at the 5-HTlA somatodendritic autoreceptor and that (S)W A Y -100135 acts as a partial agonist at this site. Aspects of tic like behaviour and serotonergic control are discussed.

Effects of some 5-hydroxytryptamine and related ligands in anxiety models

Drugs acting at 5-HT receptors were evaluated on three animal models of anxiety. On the elevated X-maze test the majority of 5-HT1 agonists were found to be anxiogenic. However, ipsapirone was anxiolytic and buspirone and gepirone were inactive. The 5-HT2 agonist DOI and the 5-HT2 antagonist ritanserin were anxiolytic while ICI 169,369, a 5-HT2 antagonist was inactive. All 5-HT3 antagonists tested were inactive in this test, while the indirect serotomimetics zimeldine and fenfluramine were anxiogenic. Neither beta-adrenoceptor agonists nor antagonists had reproducible effects on anxiety in this model. Combined beta-1/beta-2 adrenoceptor antagonists reversed the anxiogenic effects of 8-OH-DPAT while selective beta-1 or beta-2 antagonists did not. On the social interaction model the 5-HT1 agonists 8-OH-DPAT, RU 24969 and 5-MeODMT were anxiogenic and ipsapirone was anxiolytic. The 5-HT2 agonist DOI and the beta-adrenoceptor- and 5-HT- antagonist pindolol were anxiolytic, while the 5-HT2 and 5-HT3 antagonists were inactive. In the marble burying test, the 5-HT upake inhibitors zimeldine, fluvoxamine, indalpine and citalopram, the 5-HT1B/5-HT1C agonists mCPP and TFMPP and the 5-HT2/5-HT1C agonist DOI reduced marble burying without affecting locomotor activity. 5-HT1A agonists and the 5-HT2 and 5-HT3 antagonists were without effect. Lesions of the dorsal raphe nucleus reversed the anxiogenic effects of 8-OH-DPAT in the X-maze model. The implication of these results for the understanding of the pharmacology of 5-HT in anxiety is discussed.

On the modulation of the effects of some 5 - HT - related agents in axiety models

The results of an investigation into how stressors interact with the action of serotonergic agents in animal models of anxiety are presented. Water deprivation and restraint both increased plasma corticosterone concentrations and elevated 5-HT turnover. In the elevated X-maze, water deprivation had a duration-dependent "anxiolytic" effect. The effect of restraint was dependent on the duration of restraint and was to inhibit maze exploration. Water-deprivation did not influence the action of diazepam or any 5-HT1A ligand in the X-maze. Restraint switched the "anxiogenic" effect of 8-0H-DPAT to either "anxiolytic" or inactive, depending on the time after the restraint when testing was performed. The Vogel conflict test detected an "anxiolytic" "anxiolytic"V"anxiolytic""anxiolytic" effect of buspirone which was additive with "anxiolytic" effects of pindolol and propranolol. Diazepam and fluoxetine were also active, but 8-0H-DPAT, ipsapirone, gepirone and yohimbine were inactive. In the elevated X-maze, "anxiogenic" responses to picrotoxin, flumazenil, RU 24969, CGS 12066B, fluoxetine and 8-0H-DPAT were detected. Other 5-HT1A ligands were inactive. Diazepam and corticosterone had "anxiolytic" effects. Increasing light intensity did not change behaviour on the elevated X-maze, but was able to reverse the effect of 8- OH-DPAT to an "anxiolytic" action. This effect was attributed to a presynaptic mechanism, because it was abolished by pCPA. The occurence of different behaviours in different reglons of the maze was shown to be susceptible to modulation by "anxiolytic" and "anxiogenic" drugs. These results are discussed in the context of there being at least two separate 5-HT mechanisms which are involved in the control of anxiety.

An investigation into the pharmacology of tics and tic-like movements

The study of tic-like movements in mice has demonstrated close parallels both in characteristics and in pharmacology with the tics which occur in TS. Head-shakes and/or other tic-like behaviours occurring spontaneously or induced by the selective 5-HT2/1C agonist DOI, alpha-melanocyte stimulating hormone, adrenocorticotrophic hormone (1-39), thyrotropin releasing hormone, or RX336-M were blocked when tested with neuroleptics such as haloperidol and/or the alpha-2 adrenoceptor agonist clonidine. The selective dopamine D1 antagonists SCH23390 and SCH39166 dose-dependently blocked spontaneous and DOI head-shakes but the selective dopamine D2 antagonists sulpiride and raclopride were ineffective. The 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone, gepirone, MDL 73005EF and buspirone (i.p) dose-dependently blocked DOI head-shakes, pindolol blocked the inhibitory effect of 8-OH-DPAT on DOI head-shakes. Parachlorophenylalanine blocked the inhibitory effect of 8-OH-DPAT and buspirone, suggesting that the 5-HT1A receptor involved is located presynaptically. The alpha-2 adrenoceptor antagonists yohimbine, idazoxan, 1-PP and RX811059 prevented the inhibitory effect of 8-OH-DPAT on DOI head-shakes suggesting that this 5-HT1A - 5-HT2 receptor interaction is under the modulatory control of adrenoceptors. Because kynurenine has previously been found to potentiate head-shaking, plasma kynurenine concentrations were measured in seven TS patients and were significantly higher than controls, but neopterin and biopterin were unchanged. The relationship between tic-like movements in rodents and their implications for understanding the aetiology and treatment of TS is discussed.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.