Modulation of agonist binding by guanine nucleotides in CHO cells expressing muscarinic m1 – m5 receptors

In membranes prepared from CHO-m2 cells, inhibition of [³H]-N-methylscopolamine ([³H]NMS) binding by several muscarinic agonists resulted in competition curves with Hill slopes significantly different from unity. Addition of 5-guanylylimidodiphosphate (Gpp(NH)p) led to an increase in the IC₅₀ value of the agonists with significant steepening of the inhibition curves. The shift in potency induced by Gpp(NH)p differed among the agonists with a rank order of oxotremorine-M = carbachol > oxotremorine > McN-A-343 = pilocarpine. In CHO-m4 membranes, Gpp(NH)p was less efficacious than in CHO-m2 membranes whereas no effect of the guanine nucleotide was found in membranes prepared from CHO-m1, -m3, and-m5 cells. No major differences in the effect of Gpp(NH)p among agonists were found in CHO-m4 cells. Atropine binding was not affected by the guanine nucleotide. Together, these results indicate that coupling of G-proteins to muscarinic receptors linked to inhibition of cyclic adenosine monophosphate (cAMP) (m2 and m4) but not of those linked to phosphoinositol turnover (m1, m3 and m5) can be perturbed by Gpp(NH)p. The differential effects observed with Gpp(NH)p between agonist binding to m2 and m4 receptors appear to be receptor-specific and may reflect differences in the G proteins activated by these receptors in CHO cells.     

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21–41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (NΔ15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion NΔ15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (NΔ30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (NΔ41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.     

The discriminative stimulus properties of LY233708, a selective serotonin reuptake inhibitor, in the pigeon

Rationale: Understanding the contribution of the various serotonin (5-HT) receptor subtypes to the behavioral effects of selective serotonin reuptake inhibitors (SSRIs) may contribute to the discovery of increasingly effective drugs for the treatment of conditions such as depression, panic and obsessive-compulsive disorders. Objectives: A drug discrimination procedure was used to determine whether the administration of an SSRI was associated with a specific interoceptive stimulus cue and to what extent that cue was related to activation of the 5-HT_1A receptor. Method: Pigeons were trained to discriminate 20 mg/kg of the short acting, SSRI, LY233708 dihydrochloride dihydrate [(–)-cis-1-(6-chloro-1,2,3,4- tetrahydro-1-methyl-2-naphthalenyl)piperazine] from saline. Results: LY233708 induced a specific, dose-related stimulus cue. The SSRIs, fluoxetine and citalopram, induced dose-related responding on the LY233708-associated key. In contrast, nisoxetine, a selective norepinephrine uptake inhibitor, induced responding on the saline-associated key. The prototypical 5-HT_1A agonist, 8-OH-DPAT [8-hydroxy-(2-di-n-propylamino)tetralin] substituted for LY233708. This generalization was blocked by the selective 5-HT_1A antagonist, WAY-100635 [N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide]. Conclusion: These data demonstrate that an SSRI can induce a specific, stable discriminative stimulus that appears to involve activation of the 5-HT_1A receptor in the pigeon. Received: 11 January 1999 / Final version: 7 May 1999     

Serotonin (5-HT) release in the dorsal raphé and ventral hippocampus: Raphé control of somatodendritic and terminal 5-HT release

Summary Somatodendritic and terminal release of serotonin (5-HT) was investigated by simultaneously measuring extracellular concentrations of 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in the dorsal raphé and ventral hippocampus in freely moving rats. Perfusion of tetrodotoxin (TTX, 1M and 10M) into the dorsal raphé simultaneously decreased dorsal raphé and hippocampal 5-HT release. However, following TTX perfusion into the hippocampus (10M), hippocampal 5-HT release was profoundly reduced but dorsal raphé 5-HT remained unchanged.Systemic injections with the 5-HT_1a agonist, buspirone (1.0–5.0mg/kg, i.p.) decreased 5-HT and 5-HIAA and increased HVA concentrations in the dorsal raphé and in the hippocampus. The decreases in raphé and hippocampal 5-HT induced by systemic buspirone were antagonized in rats pretreated with 1.OmM (–) pindolol, locally perfused into the dorsal raphé. Local dorsal raphé perfusion of (–) pindolol alone (0.01–1.0mM) increased dorsal raphé 5-HT and concomitantly induced a small increase in hippocampal 5-HT. Buspirone perfusion into the dorsal raphé did not change (10 nM, 100nM), or produced a small increase (1.0mM) in raphé 5-HT, without changing hippocampal 5-HT.These data provide evidence that 5-HT release in the dorsal raphé is dependent on the opening of fast activated sodium channels and that dorsal raphé 5-HT_1a receptors control somatodendritic and hippocampal 5-HT release.     

Effect of repeated administration of dopamine agonists on striatal neuropeptide mRNA expression in rats with a unilateral nigral 6-hydroxydopamine lesion

Summary Striatal mRNA expression for preproenkephalin (PPE) and preprotachykinin (PPT) was studied in unilateral 6-OHDA lesioned rats treated subchronically with a range of selective and non-selective D-1 or D-2 dopamine (DA) agonists. Apomorphine (5mg/kg sc), pergolide (0.5mg/kg sc), SKF 38393 (5mg/kg sc), SKF 80723 (1.5mg/kg sc), and quinpirole (5mg/ kg sc), or 0.9% saline (150l sc) were all given twice daily (except pergolide: once daily) for 7 days. The abundance of PPE mRNA was not altered by any of these DA agonists in the intact striatum contralateral to the 6-OHDA lesion. Only apomorphine and quinpirole increased the abundance of PPT mRNA in the intact striatum. In saline treated 6-OHDA lesioned animals PPE mRNA was elevated (+160%, p < 0.005) and PPT mRNA decreased (–36%, p < 0.005) in the denervated striatum. The up-regulation of striatal PPE mRNA in the lesioned striatum was reversed only by pergolide. The downregulation of striatal PPT mRNA in the lesioned striatum was reversed only by apomorphine. The differential sensitivity of the striatal PPE message to the long-acting DA agonist pergolide, and of the striatal PPT message to the mixed D-1/D-2 DA agonist apomorphine suggests that the striatopallidal enkephalinergic pathways are mainly regulated by prolonged DA receptor stimulation, whereas the striatonigral substance P pathways are mainly regulated by mixed D-1/D-2 DA receptor stimulation.     

Promotion of cell growth by stimulation of cloned human 5-HT1D receptor sites in transfected C6-glial cells is highly sensitive to intrinsic activity at 5-HT1D receptors

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT_1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT_1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144–156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC₅₀: 25 nM) promoted cell growth of C6-glial/5-HT_1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC₅₀ in nM): zolmitriptan (0.41), 2-methyl-4-(5-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127,935; 0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-y)]ethylamine (MK-462; 3.0), and R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC₅₀-values correspond to the compounds binding affinities at the human 5-HT_1D receptor site and, with the exception of GR 127,935 and metergoline, also to the EC₅₀-values found by measuring over 5 min inhibition of forskolin (100 M)-stimulated cAMP formation. Prolonged exposure of GR 127,935 (3 h) and metergoline (30 min) to cells yielded EC₅₀ values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonists methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated CAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT_1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 M) but not by spiperone (1 M). Micromolar concentrations of the 5-HT_1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3, 2-b]pyril-5-one (CP 93,129) and of the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT_1D receptor site. These effects were sensitive to ketanserin (1 M) antagonism, but not to blockade by -adrenergic blockers and the 5-HT₂ receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT_1A, 5-HT_1B and 5-HT₂ receptors are not implicated in 5-HT-stimulated C6-glial/5-HT_1D cell growth. In conclusion, human 5-HT_1D receptors are involved in the growth of C6-glial/5-HT_1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT_1D receptors.     

SPECT imaging of dopamine and serotonin transporters with [123I]β-CIT. Binding kinetics in the human brain

Summary Single photon emission computerized tomography (SPECT) studies in non-human primates have previously shown that the cocaine derivative [¹²³I]-2--carbomethoxy-3--(4-iodophenyl)-tropane ([¹²³I]-CIT) labels dopamine transporters in the striatum and serotonin transporters in the hypothalamusmidbrain area. Here, we report on the regional kinetic uptake of [¹²³I]-CIT in the brain of 4 normal volunteers and 2 patients with Parkinson's disease. In healthy subjects striatal activity increased slowly to reach peak values at about 20 hours post injection. In the hypothalamus-midbrain area peak activities were observed at about 4 hours with a slow decrease thereafter. Low activity was observed in cortical and cerebellar areas. The striatal to cerebellar ratio was about 4 after 5 hours and 9 after 20 hours. In 2 patients with idiopathic Parkinson's disease striatal activity was markedly decreased while the activity in hypothalamus-midbrain areas was only mildly diminished. Uptake into cortical and cerebellar areas appeared to be unchanged in Parkinson's disease. Consequently, in Parkinson's disease the striatal to cerebellar ratio was decreased to values around 2.5 after 20 hours. These preliminary methodological studies suggest that [¹²³I]-CIT is a useful SPECT ligand for studying dopamine and possibly also serotonin transporters in the living human brain.     

An examination of factors influencing adrenergic transmission in the pithed rat,with special reference to noradrenaline uptake mechanisms and post-junctional α-adrenoceptors

Summary Adrenergic mechanisms were analysed in the pithed rat to determine to what extent the actions of drugs observed in vitro are relevant in situ.The drugs examined were those which are known to block the neuronal or extraneuronal uptake of noradrenaline (cocaine, desipramine and corticosterone) or to be antagonists at post and/or pre-junctional -adrenoceptors (prazosin and yohimbine) together with the antidepressant, mianserin, which has been implicated in several of these actions. These drugs were tested against the arterial diastolic pressor, cardiac chronotropic and vas deferens responses to sympathetic nerve stimulation, to indirect sympathomimetics or to direct sympathomimetics, which were chosen according to whether they were substrates for noradrenaline uptake processes or selective between adrenoceptors.Pressor and cardiac responses to sympathetic nerve stimulation or to intravenous noradrenaline were potentiated by blockade of neuronal uptake but only the pressor effect of noradrenaline was potentiated by blockade of extraneuronal uptake. The effects of the antagonists suggested that the pressor effects of noradrenaline and of sympathetic nerve stimulation result from a combination of activation of ₁- and ₂-adrenoceptors, but that the effect of noradrenaline had a relatively greater contribution from the ₂-adrenoceptors. Mianserin was found to potentiate adrenergic responses at low doses, to produce limited antagonism at post-junctional ₁-adrenoceptors in high doses but to have no detectable effect at postjunctional ₂-adrenoceptors.     

β-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: Dissociation of stimulation and blockade

Summary The cardiac stimulant actions of nine -adrenoceptor blocking agents were examined in kitten papillary muscles and in isolated atria of kittens and guinea pigs to determine to what extent these drugs behaved as classical partial agonists. In many ways the agents do appear to comprise a spectrum of partial agonists with widely differing efficacies. However, in one respect the actions of some of the -blockers did not fit into the classical mold. Several -blockers were found to exert stimulant effects only in concentrations appreciably higher than those required for substantial -adrenoceptor blockade. These observations suggest that more than one type of -adrenoceptor may be involved in the production of sympathomimetic effects on cardiac muscle.     

Profil psychopharmacologique d'un nouvel agoniste Dopaminergique,le RU 24213

The psychopharmacological properties of RU 24213 were compared to those of other dopaminergic agonists (apomorphine, dexamphetamine, bromocriptine and l-dopa) in various behavioural tests. In naive mice the drug reduced the locomotor hyperactivity in the primary exploratory phase and produced stimulation in the subsequent stabilized activity period. In rats it provoked dose-related stereotypies, specially gnawing and sniffing. It delayed the cataleptic state induced by prochlorperazine without affecting its intensity. In animals unilaterally lesioned with 6-OHDA in the nigro-striatal pathway, RU 24213 caused contralateral turning. It exhibited relatively weak emetic and anorexic effects in dogs. Core temperature recordings in rats revealed a biphasic hypo- and hyperthermic activity. In drug interaction studies it was observed that stereotypies and rotations induced by RU 24213 were blocked by haloperidol and decreased by MT. Reserpine respectively decreased stereotypies and increased ipsilateral rotations in rats with unilateral electrolytical striatal lesion.The results obtained suggest that RU 24213 stimulates dopamine receptors both directly and indirectly. In this respect it could be compared to bromocriptine but unlike this latter compound it has an immediate effect which is of shorter duration.