5-hydroxytryptamine4 receptor agonists facilitate cholinergic transmission in the circular muscle of guinea pig ileum: antagonism by tropisetron and DAU 6285.

The effect of 5-hydroxytryptamine (5-HT), BIMU 8 (endo-N-(8-methyl-8-azabicyclo [3.2.1.] oct-3-yl)-2,3-dihydro-3-(1-methyl)ethyl-2-oxo-1H-benzimidazole-1- carboxamide hydrochloride) and cisapride was studied on the electrically-induced neurogenic cholinergic twitch contractions in the guinea pig ileum circular muscle. These compounds caused a concentration-dependent increase in the amplitude of submaximal twitch contractions with the following rank order of potency: 5-HT greater than BIMU 8 = cisapride. The effect of 5-HT was competitively antagonized by tropisetron (ICS 205-930) (apparent pA2 value: 6.4), suggesting an interaction at 5-hydroxytryptamine4 (5-HT4) receptors. The novel benzimidazolone derivative DAU 6285 (endo-6-methoxy-8-methyl-8-azabicyclo [3.2.1.] oct-3-yl-2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylate hydrochloride), antagonized the effect of 5-HT, BIMU 8 and cisapride with apparent pA2 values in the range 7.1-7.3. Our findings demonstrate that cholinergic neurones innervating the circular coat are endowed with excitatory 5-HT4 receptors. DAU 6285 is approximately 5-9-fold more potent than tropisetron as antagonist at these receptors.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Serotonin induced vasodilatation in the human forearm is antagonized by the selective 5-HT3 receptor antagonist ICS 205-930

The role of 5-HT3 receptors in the biphasic vasodilator response to serotonin (5-hydroxytryptamine; 5-HT) was investigated in the forearm of 7 young healthy volunteers (aged 22-32 years). Single dose infusions of 5-HT (1 ng/kg/min) and of acetylcholine (ACh, 500 ng/kg/min) were administered into the brachial artery. Subsequently combined infusions of 5-HT together with the selective 5-HT3 receptor antagonist ICS 205-930 (350 and 700 ng/kg/min), and ACh together with ICS 205-930 (700 ng/kg/min) were given. After a pause of at least 1 hour the single infusions of 5-HT and ACh were repeated. Subsequently, 5-HT and ACh were infused together with atropine (100 ng/kg/min). Forearm blood flow (FBF) was measured by R-wave triggered venous occlusion plethysmography. Heart rate (HR) and i.a. blood pressure (BP) were recorded semi-continuously. None of the drugs in the doses used did induce systemic hemodynamic effects. After an initial rapid transient increase in FBF of 316 +/- 55%, 5-HT elicited a persistent increase in FBF of 90 +/- 22% (mean +/- SEM, p less than 0.05 for both). ACh induced a monophasic vasodilatation of 475 +/- 123% (p less than 0.05). Both the initial transient and the persistent dilatator response to 5-HT were attenuated by ICS 205-930 350 ng/kg/min (p = 0.057, n = 5) and 700 ng/kg/min (p less than 0.05, n = 7). The highest dose of ICS 205-930 did not significantly influence the dilatator response to ACh. Atropine abolished the ACh induced vasodilatation (p less than 0.05), but did not influence the biphasic dilatator response to 5-HT. Thus the 5-HT induced biphasic vasodilatation was antagonized by ICS 205-930, indicating that this response was mediated by 5-HT3 receptor activation. The fact that atropine did not influence the vascular response to 5-HT suggests that 5-HT did not induce vascular relaxation indirectly by the release of ACh from cholinergic nerve endings.     

The 5-HT3 antagonist tropisetron (ICS 205-930) is a potent and selective alpha7 nicotinic receptor partial agonist

The 5-HT3 receptor antagonist tropisetron (ICS 205-930) was found to be a potent and selective partial agonist at alpha7 nicotinic receptors. Two other 5-HT3 receptor antagonists, ondansetron and LY-278,584, were found to lack high affinity at the alpha7 nicotinic receptor. Quinuclidine analogues (1 and 2) of tropisetron were also found to be potent and selective partial agonists at alpha7 nicotinic receptors.     

Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells.

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN'-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [( 3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.     

5-Hydroxytryptamine affects rat migrating myoelectric complexes through different receptor subtypes: evidence from 5-hydroxytryptophan administration

The effect of the serotonin precursor 5-hydroxytryptophan (5-HTP) on jejunal migrating myoelectric complexes (MMCs) was investigated in conscious rats. Subcutaneous administration of low doses of 5-HTP (1-2 mg/kg) shortened the period between migrating complexes, whereas high doses of the compound (4-8 mg/kg) disrupted the MMC pattern. The serotonin (5-HT2) antagonist methysergide (8 mg/kg s.c.) did not alter basal MMC, neither did it prevent the effect of a low dose of 5-HTP; conversely, it antagonized the disruption due to the high dose. The 5-HT3 antagonist ICS 205-930 (30 micrograms/kg s.c.) decreased MMC frequency; administration of 2 mg/kg 5-HTP following ICS 205-930 brought the frequency of myoelectric complexes back to basal values. Both effects of 5-HTP were prevented by the decarboxylase inhibitor benserazide (85 mg/kg i.p.), which per se caused a transient inhibition of spiking activity. The results suggest that rat MMCs can be influenced in a composite fashion by progressively increasing concentrations of 5-HT, which in turn activate different receptor subtypes. A peripheral neuronal receptor, probably belonging to the 5-HT3 subclass, mediates the increase in MMC frequency observed after low doses of 5-HTP; higher levels of serotonin activate 5-HT2 receptors, causing disruption of cycling activity. Additionally, 5-HT3 receptors, but not 5-HT2, appear to be relevant for the regulation of the MMC pattern by the endogenous amine.     

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.     

Detection and Characterization of the Serotonin 5-HT1D Receptor in Rat and Human Brain

In the presence of 1 microM ( +/- )-pindolol [to block 5-hydroxytryptamine (5-HT, serotonin) 5-HT 1A and 5-HT 1B receptors] and 100 nM mesulergine (to block 5-HT 1C receptors), 2.0 nM [3H]5-HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5-HT binding, in the presence of 1 microM ( +/- )-pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5-HT receptor subtypes. Compounds with high affinity for 5-HT 1A (8-hydroxydipropylaminotetralin), 5-HT 1B (trifluoromethylphenylpiperazine), 5-HT 1C (mesulergine), 5-HT 2 (4-bromo-2,5-dimethoxyphenylisopropylamine), and 5-HT3 (ICS 205-930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5-HT binding site in rat and human brain tissue. This putative novel 5-HT receptor has a similar pharmacology to the "5-HT 1D" site detected in bovine brain by Heuring and Peroutka.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.     

Activation of peripheral serotonin2 receptors induces hypothermia in mice

The effects of peripherally administered serotonin (5-HT) on the rectal temperature were investigated. 5-HT i.p. induced a dose-dependent hypothermia in mice. The hypothermic effects of 5-HT were strongly antagonized by the 5-HT1 and 5-HT2 receptor antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. However, the 5-HT1 receptor antagonist pindolol and the 5-HT3 receptor antagonist ICS 205-930 were without effect. In addition, the peripheral 5-HT2 receptor antagonist xylamidine strongly reduced 5-HT-induced hypothermia. These results indicate that the activation of the peripheral 5-HT2 receptors induces hypothermia, although the central 5-HT2 receptors have been suggested to relate to hyperthermia.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

5-HT3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line

Serotonin (5-HT) induces a large inward current accompanied by a conductance increase when applied focally to either neurons of mouse hippocampal cultures or cells of the NG108-15 clonal cell line. In both systems, the response is blocked by ICS 205-930, curare, and metoclopramide, while 2-methyl-5-HT is an agonist. The actions of ICS 205-930 and 2-methyl-5-HT indicate that the response is mediated by the 5-HT3 receptor. In NG108-15 cells the response activated in as little as 35 ms. The rapidity of the response suggests a direct coupling between the 5-HT3 receptor and a channel, which are probably both part of a single membrane protein. In both cell types, prolonged application of 5-HT resulted in desensitization; the rates of desensitization were accelerated by the adenylate cyclase activator forskolin. The 5-HT3 receptor has much in common with the nicotinic receptor and is probably involved in rapid synaptic transmission in the mammalian brain. Since this response is modulated by manipulations that elevate intracellular cAMP levels, the central synapses in which this receptor operates may exhibit plasticity.     

Serotonin-induced hypoglycemia and increased serum insulin levels in mice

The effects of serotonin (5-HT) on plasma glucose levels were studied. 5-HT above 20 mg/kg induced apparent hypoglycemia in mice. The hypoglycemic effects of 5-HT were strongly antagonized by methysergide but only partially inhibited by ketanserin. However, ICS 205-930 was without effect. This indicates that the hypoglycemia induced by 5-HT is mediated by both the 5-HT1 and 5-HT2 receptors. 5-HT also produced an increase in serum immunoreactive insulin (IRI) which was completely inhibited by methysergide and partially antagonized by ketanserin. It is suggested that the 5-HT-induced increase in IRI is elicited by the activation of the 5-HT1 and 5-HT2 receptors, which is similar to the results obtained with plasma glucose. These results indicate that the 5-HT receptors may regulate blood glucose levels by modifying the release of insulin.     

The pharmacology of the isolated foregut of the locust Schistocerca gregaria—II. characterization of a 5-HT2-like receptor

1. 5-HT (10⁻⁸−5 × 10⁻⁶ M) relaxed isolated locust foreguts.2. The effects of 5-HT were mimicked by 5,6-DHT, 5-MeOT, tryptamine, 5-MeT, MK212 and methysergide while 5-hydroxyindole, 5-hydroxyindole acetic acid, 5-hydroxytryptophol, NN-DMT, 8-OH DPAT and RU 24969 were without effect.3. Ketanserin (pA₂ = 5.65) was a competitive antagonist of the effects of 5-HT, MK212 and methysergide.4. Mianserin (pA₂ = 6.3) was also a competititve antagonist of 5-HT but ICS 205-930 had no antagonistic effect on 5-HT-induced relaxation.5. It is concluded that 5-HT relaxes the locust foregut by interacting with 5-HT₂-like receptors.     

Serotonin Stimulation of 5-HT4 Receptors Indirectly Enhances In Vivo Dopamine Release in the Rat Striatum

Abstract: Serotonin (5-HT) applied at 1, 3, and 10 µ M into the striatum of halothane-anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5-HT effect was partially reduced by 1 or 10 µ M GR 125,487, a 5-HT₄ antagonist, and by 100 µ M DAU 6285, a 5-HT_3/4 antagonist, whereas the 5-HT_1/2/6 antagonist methiothepin (50 µ M ) was ineffective. In the presence of tetrodotoxin the effect of 1 µ M 5-HT was not affected by 5-HT₄ antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5-HT₄ agonist ( S )-zacopride (100 µ M ). In striatal synaptosomes, 1 and 10 µ M 5-HT increased the outflow of newly synthesized [³H]DA up to 163 and 635% of control values, respectively. The 5-HT₄ agonists BIMU 8 and ( S )-zacopride (1 and 10 µ M ) failed to modify [³H]DA outflow, whereas 5-methoxytryptamine (5-MeOT) at 10 µ M increased it (62%). In prelabeled [³H]DA synaptosomes, 1 µ M 5-HT, but not ( S )-zacopride (1 and 10 µ M ), increased [³H]DA outflow. DAU 6285 (10 µ M ) failed to modify the enhancement of newly synthesized [³H]DA outflow induced by 5-MeOT or 5-HT (1 µ M ), whereas the effect of 5-HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 µ M ) alone or in the presence of DAU 6285. These results show that striatal 5-HT₄ receptors are involved in the 5-HT-induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.     

Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [H]GR65630 to rat cortical membranes

In the absence of detergent, specific binding of [³H]GR65630, a 5-hydroxytryptamine₃ (5-HT₃) antagonist, determined in the presence of 5-HT₃ receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [³H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X-100) decreased nonspecific binding, increasing the proportion of specific binding to 70% of total binding. In the presence of 0.1% Triton X-100, binding of [³H]GR65630 was rapid, reversible and saturable at 25°C. The rank order of 5-HT₃ receptor active drugs in inhibiting [³H]GR65630 binding was quipazine > ICS205-930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [³H]GR65630 efficiently labeled 5-HT₃ receptors in the presence of Triton X-100. Triton X-100 improved 5-HT₃ receptor binding with rat brain membranes.     

The design and pharmacology of novel selective muscarinic agonists and antagonists

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.     

Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.     

The relaxing effect of SCH 23390 in the molluscan smooth muscle

1. SCH 23390 (SCH, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) produced the relaxation of ACh-induced contraction in the anterior byssus retractor muscle of Mytilus edulis (ABRM) in a dose-dependent manner between 10⁻⁹-10⁻⁶M.2. The dose-response curve of SCH was shifted in parallel to the right by ketanserin (KET) with pA₂ value of 5.14 ± 0.08 and by 1-(1-naphthyl)piperazine (NAP) with that of 5.06 ± 0.01, but not by cyproheptadine (CYP), mianserin (MIA), butaclamol (BUTA), ICS 205–930 (ICS) and MDL 72222 (MDL) at 3 × 10⁻⁵ M.3. α-Methyl-serotonin (α-Me-5-HT), a selective 5-HT₂ receptor agonist dose-dependently relaxed the ACh-induced contraction of ABRM. The dose-response curve of α-Me-5-HT was shifted in parallel to the right by KET with pA₂ value of 5.01 ± 0.02, but not by BUTA, CYP, MIA, ICS and MDL at 3 × 10⁻⁵ M.4. These findings suggest that 5-HT₂-like receptors exist in the ABRM, and that the relaxation induced by SCH is mediated through these receptors.